Fatty alcohol forming acyl reductases (FARs) and methods of use thereof

ABSTRACT

The present disclosure provides methods useful for producing fatty alcohol compositions from recombinant host cells. The disclosure further provides variant fatty acyl-CoA reductase (FAR) enzymes, polynucleotides encoding the variant FAR enzymes, and vectors and host cells comprising the same.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/171,138, filed Jun. 28, 2011, now issued as U.S. Pat. No. 8,574,878,which claims benefit of priority of U.S. Provisional Application No.61/359,211, filed Jun. 28, 2010, the entire content of each of which isincorporated herein by reference.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 90834-888301_ST 25.TXT, created onNov. 20, 2013, 51,866 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Reliance on petroleum-derived fuels has depleted the supply of naturalresources and has required increased reliance on imported gasoline anddiesel products. In addition, the burning of petroleum-based fuels hasincreased the amount of greenhouse gasses (e.g., carbon dioxide andmethane) in the atmosphere that is contributing to the gradual warmingof the earth's climate.

Fuels, such as biodiesel, that are made from animal or vegetableproducts burn cleaner than petroleum-derived fuels and do not produce anet increase in greenhouse gases. Furthermore, they are a sustainableenergy source and have the potential to reduce the United States'reliance on imported petroleum-based products. However, there is aconcern that using land to produce fuel crops rather than food cropswill contribute to world hunger.

Fatty acids are the principal component of cell membranes and are usedby nearly all organisms as a primary source of energy storage. Fattyalcohols are the reduction products of fatty acids and, like fattyacids, can be produced enzymatically by cultured cells. Fatty alcoholscan be reacted with acids to form ester compositions similar to thosepresent in biodiesel fuel, or reduced to form kerosene-likecompositions, or hydrocarbon compositions similar to petrodiesel.Enzymes that convert fatty acyl-thioester substrates (e.g., fattyacyl-CoA or fatty acyl-ACP) to fatty alcohols are commonly referred toas fatty alcohol forming acyl-CoA reductases or fatty acyl reductases(“FARs”).

PCT Publication No. WO 2007/136762 discloses genetically engineeredmicroorganisms for the production of fatty acid derivatives and methodsof their use.

U.S. Pat. No. 5,370,996 and Metz et al., 2000, Plant Physiology122:635-644 disclose isolation and characterization of a fatty acylreductase (FAR) enzyme from the desert shrub Simmondsia chinensis, morecommonly known as jojoba.

Moto et al., 2003, Proc. Nat'l Acad. Sci USA 100(16):9156-9161 disclosesthe isolation and characterization of a FAR enzyme from the silk mothBombyx mori.

Reiser et al., 1997, J. Bacteriol. 179(9):2969-2975 discloses theisolation and characterization of a fatty acyl CoA reductase enzyme fromthe wax ester producing bacterium Acinetobacter calcoaceticus thatreduces a fatty acyl-CoA substrate with chain lengths from C14 to C22 tothe corresponding fatty aldehyde, requiring a dehydrogenase enzyme forconversion of the fatty aldehyde to the fatty alcohol.

In theory, these FAR enzymes could be expressed in heterologous hosts asa means of producing a non-petroleum-based, renewable source of fattyalcohol or derivative compositions for use in biofuels. However, whenexpressed in heterologous hosts such as E. coli and yeast, the yields offatty alcohols obtained are insufficient for certain applications. Inaddition, at most, only a small fraction of fatty alcohols produced aresecreted by the microorganisms, increasing substantially the cost ofpurification.

Accordingly, there is a need in the art for enzymes such as FAR enzymesthat can be used efficiently to produce fatty alcohols for use inindustrial applications such as but not limited to applications in thechemical industry (e.g., household detergents, including laundrydetergents in liquid and powder form, hard surface cleaners, dishwashingliquids, and the like; lubricants and solvents; industrial cleaners; andsurfactants), in the food industry, in the personal care industry (e.g.,in soaps, cosmetics, shampoos, and gels), in the medical industry, andin the fuels industry (e.g., in diesel fuels).

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides recombinant fatty alcohol formingacyl-CoA reductase (FAR) variants that exhibit improved properties. Insome embodiments, a FAR variant is capable of producing at least about1.5 times more fatty alcohol than a wild-type FAR polypeptide (e.g., aFAR polypeptide comprising SEQ ID NO:2 or SEQ ID NO:5). In someembodiments, a FAR variant is capable of producing an improved fattyalcohol profile as compared to a wild-type FAR polypeptide. In someembodiments, the invention relates to improved fatty alcohol formingacyl-CoA reductase (FAR) polypeptides capable of producing at leastabout 1.5 times more fatty alcohol than a wild-type FAR polypeptidecomprising SEQ ID NO:2 and/or producing an improved fatty alcoholprofile as compared to a wild-type FAR polypeptide comprising SEQ IDNO:2 when assayed under the same conditions. In some embodiments, theimproved fatty alcohol forming acyl-CoA reductase (FAR) polypeptides arecapable of producing at least about 1.5 times more fatty alcohol than awild-type FAR polypeptide comprising SEQ ID NO:5 and/or producing animproved fatty alcohol profile as compared to a wild-type FARpolypeptide comprising SEQ ID NO:5 when assayed under the sameconditions. In some embodiments a cell (e.g., E. coli) expressing theimproved FAR produces at least 2-fold, at least 3-fold, at least 4-fold,at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, atleast 9-fold, or at least 10-fold more fatty alcohol than a wild-typeFAR polypeptide (e.g., a FAR polypeptide comprising SEQ ID NO:2 or SEQID NO:5).

In some embodiments, the amount and/or profile of fatty alcoholsproduced is measured by determining and/or quantifying the fattyalcohols produced by a microorganism that expresses a FAR polypeptide(e.g., a wild-type FAR polypeptide or a FAR variant as described here).In some embodiments, the microorganism is a bacteria (e.g., E. coli) ora yeast (e.g., a Yarrowia or a Saccharomyces cerevisiae).

In some embodiments, the invention relates to a fatty alcohol formingacyl-CoA reductase (FAR) variant that has at least 70% (or at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%) sequence identity to SEQ IDNO:2, wherein the variant comprises a substitution at one or morepositions selected from position 134, position 138, position 511,position 510, position 2, position 140, position 421, and position 458,wherein the position is numbered with reference to SEQ ID NO:2, andwherein a cell in which the FAR variant is expressed produces more fattyalcohol than a corresponding cell of the same type in which thewild-type FAR from which the FAR variant is derived is expressed. Insome embodiments, the cell expressing the FAR variant produces at least1.5, at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, or at least 10 times more fatty alcoholthan the corresponding cell of the same type which expresses thewild-type FAR from which the FAR variant is derived. In someembodiments, the cell is a bacteria cell (e.g., E. coli). In someembodiments the cell is a yeast cell (e.g., a Yarrowia or aSaccharomyces cerevisiae cell). In some embodiments, the wild-type FARis Marinobacter algicola FAR having the amino acid sequence of SEQ IDNO:2. In some embodiments, the wild-type FAR is Marinobacter aquaeoleiFAR having the amino acid sequence of SEQ ID NO:5.

In some embodiments, the invention relates to a fatty alcohol formingacyl-CoA reductase (FAR) variant that has at least 70% (or at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%) sequence identity to SEQ IDNO:2, wherein the variant comprises a substitution at one or morepositions selected from position 14, position 18, position 63, position65, position 104, position 128, position 134, position 174, position177, position 224, position 226, position 227, position 244, position283, position 306, position 351, position 364, position 365, position370, position 376, position 377, position 389, position 404, position406, position 433, or position 487, and wherein a cell in which the FARvariant is expressed produces more fatty alcohol than a correspondingcell of the same type in which the wild-type FAR from which the FARvariant is derived is expressed. In some embodiments, the cellexpressing the FAR variant produces at least 1.5, at least 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, or at least 10 times more fatty alcohol than the corresponding cellof the same type which expresses the wild-type FAR from which the FARvariant is derived. In some embodiments, the cell is a yeast cell (e.g.,a Yarrowia or a Saccharomyces cerevisiae) or a bacterial cell (e.g., E.coli). In some embodiments, the wild-type FAR is Marinobacter algicolaFAR having the amino acid sequence of SEQ ID NO:2. In some embodiments,the wild-type FAR is Marinobacter aquaeolei FAR having the amino acidsequence of SEQ ID NO:5.

In some embodiments, a FAR variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 or SEQ ID NO:5 and comprises an amino acid substitution at oneor more positions selected from A2, T3, Q4, Q5, Q6, Q7, N8, G9, A10,A12, G14, E17, Q18, K22, V24, L33, I42, G50, L54, R60, H61, P62, A63,R65, L69, E71, A73, S74, S76, V77, H83, E87, T91, L93, H98, T101, G102,V104, S107, G110, L111, T112, P113, R115, R117, A120, G121, Q122, A125,N128, S132, N134, E137, E138, D140, A142, K144, L148, E151, V153, N160,A162, N174, N177, Q180, V185, 1186, P188, T197, D198, E202, E204, E205,V207, L209, D212, K213, V217, R220, K224, L226, E227, K229, R236, E237,S244, D245, T246, L257, K260, A261, S263, G264, S266, 1269, S283, 1287,E288, V290, A295, A299, E303, V305, S306, V318, 1328, L330, S331, L332,A333, 5339, G340, Q341, R342, G350, G351, K359, L364, M365, A366, T370,A374, D376, Q377, Y380, R381, T384, A389, D396, V397, V398, V399, G400,G401, R403, V404, P405, L406, A409, G410, A412, M413, A416, Q418, E421,N427K, D429, T430, 8432, S433, T436, 1437, F440, A443, P444, Y446, S452,S458, R459, L463, D464, V466, A472, Q474, L479, I484, G487, N490, E496,K498, L499, Y500, S501, L502, A504, A505, D506, T507, R508, K509, K510,A511, and A512, wherein the position is numbered with reference to SEQID NO:2. In some embodiments, the variant comprises one or more aminoacid substitutions selected from A2D/F/G/H/I/P/N/Q/T/V/W, T3R, Q4R, Q5S,Q6P, Q7N, N8K/S, G9D/F, A10T, A12T/V, G14N/R/V/W, E17D, Q18I, K22E,V241, L33V, I42L, G50S/V, L54P, R60H, H61R, P62S, A63R/Y, R65G/Q/Y,L69E/Q, E71K, A73K/V, S74K/P, S76K/N/R, V77A/I, H83R, E87G/V, T91I/R,L93V, H98P/R, T101L, G102C, V104I/M, S107C/L/W, G110D, L111S, T112A,P113D/L, R115A/H, R117D, A120V, G121H/S, Q122R, A125V, N128H, S132G,N134K/R/S, E137L, E138L/Q, D140C, A142V, K144Q, L148E, E151L, V153I,N160S, A162T, N174C, N177Q/R/T, Q180H/R, V185A/I, I186A/GN, P188A/I/M/S,T197P, D198Q, E202G, E204G, E205G/P, V2071/L, L209K/N, D212R, K213R,V217L, R220C, K224R, L226A/M, E227A/G/H/R/T, K229R, R236K, E237L,S244A/F/G/H/P, D245N, T246A, L257K, K260R/T, A261D, S263P, G264S, S266A,I269T, S283E/F/K/M/T/V, 1287L, E288Q, V290I, A295T/V, A299T, E303G,V3051, S306F/H/N/W, V3181, 1328T, L330V, S331V, L332S, A333T, S339G/V,G340P/S/V, Q341K, R342L, G350S, G351C, K359L, L364F/I, M365N, A366T/V,T370A/I, A374K/Y, D376P, Q377C/K/Y, Y380K/N/R, R381C, T384R,A389I/L/M/V, D396G, V3971/L, V398Y, V399T, G400A/L, G401A/C/I/L/S/V,R403C/S, V404A, P405A/C/F/G/L/S/V/W, L406Y, A409V/W/Y,G410A/C/H/N/Q/R/S, A412C/F/M/V, M413L/R, A416L/V, Q418I/R/V/Y,E421I/L/N/P/R/S/V/Y, N427K, D429E/K/N/Q/R, T430H/I/R, R432C/Q,S433F/H/K/L/N/W, T436D/K/Q, I437V, F440L, A443T, P444S, Y446H,S452A/G/N, S458G/L/M/Q, R459H, L463E/T, D464G, V466E/Q/R, A472V, Q474R,L479Q, I484V, G487R/S/T/Y, N490S, E496A, K498A, L499A/H/I/N/P/R/S,Y500C/G/H/L/N/P/Q/R/S/W, S501 G/R, L502A/P/Q/R/S, A504G/R, A505K,D506G/S, T507A/G/P/R/S, R508D/G/H, K509D/E/G/H/N/R/S/Y, K510A/D/G/P/S/Y,A511G/I/K/P/Q/R/S/T, and A512K/S/T.

In some embodiments, a variant as described herein is encoded by apolynucleotide that hybridizes at high stringency to the complement ofSEQ ID NOs: 1, 3, 4, 13, or 14 and comprises one or more amino acidsubstitutions as described herein.

In some embodiments, the variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 or SEQ ID NO:5 and comprises an amino acid substitution relativeto SEQ ID NO:2 at one or more positions selected from position 134,position 138, position 188, position 458, and position 511, wherein theposition is numbered with reference to SEQ ID NO:2. In some embodiments,the variant comprises an amino acid substitution at one or morepositions selected from N134, E138, P188, S458, and A511. In someembodiments, the variant comprises one or more amino acid substitutionsselected from N134R, E1380, P188S, S458Q, and A511T.

In some embodiments, the variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 or SEQ ID NO:5 and comprises an amino acid substitution relativeto SEQ ID NO:2 at one or more positions selected from position 303,position 401, position 405, position 412, position 416, position 418,position 458, position 502, position 508, and position 509, wherein theposition is numbered with reference to SEQ ID NO:2. In some embodiments,the variant comprises an amino acid substitution at one or morepositions selected from E303, G401, P405, A412, A416, Q418, S458, L502,R508, and K509. In some embodiments, the variant comprises one or moreamino acid substitutions selected from E303G, G401A/L/S/V, P405A/C/F/UV,A412V, A416L, Q418I/V, S458Q, L502S, R508G/H, and K509D/H.

In some embodiments, the variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 or SEQ ID NO:5 and comprises an amino acid substitution relativeto SEQ ID NO:2 at one or more positions selected from position 22,position 87, position 209, position 264, position 401, position 416,position 500, position 508, and position 509, wherein the position isnumbered with reference to SEQ ID NO:2. In some embodiments, the variantcomprises an amino acid substitution at one or more positions selectedfrom K22, E87, L209, G264, G401, A416, Y500, R508, and K509. In someembodiments, the variant comprises one or more amino acid substitutionsselected from K22R, E87G, L209K/L/N, G264S, G401V, A416L, Y500D,R508D/G, and K509D/H/N/Y.

In some embodiments, the variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 or SEQ ID NO:5 and comprises an amino acid substitution relativeto SEQ ID NO:2 at one or more positions selected from N134, E138, P188,P405, Q418, and A511, wherein the position is numbered with reference toSEQ ID NO:2. In some embodiments, the variant comprises one or moreamino acid substitutions selected from N134S, E138Q, P188S, P405V,Q418V, and A511T. In some embodiments, the variant has the amino acidsequence of SEQ ID NO:6, the sequence of Variant 370 described in Table2.

In some embodiments, the variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 or SEQ ID NO:5 and comprises an amino acid substitution relativeto SEQ ID NO:2 at one or more positions selected from N134, E138, P188,L209, P405, Q418, S458, L502, R508, K509, and A511, wherein the positionis numbered with reference to SEQ ID NO:2. In some embodiments, thevariant comprises one or more amino acid substitutions selected fromN134S, E138Q, P188S, L209K, P405V, Q418V, S458Q, L502S, R508D, K509D,and A511T. In some embodiments, the variant has the amino acid sequenceof SEQ ID NO:7, the sequence of Variant 391 described in Table 2. Insome embodiments, the variant comprises at least about 70% (or at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%) sequence identity to theamino acid sequence of SEQ ID NO:7 and further comprises one or moreamino acid substitutions selected from G14R/V, Q18I, A63R, R65G,V104I/M, N128H, S134R, N174C, N177T, K224R, L226M, E227R, S244P,S283F/M, S306W, G351C, L364I, M365N, T370I, D376P, Q377K, A389I, V404A,L406Y, S433K, or G487R. In some embodiments, the variant comprises theamino acid sequence of SEQ ID NO:7 and further comprises one or moreamino acid substitutions selected from G14R/V, Q18I, A63R, R65G,V104I/M, N128H, S134R, N174C, N177T, K224R, L226M, E227R, S244P,S283F/M, S306W, G351C, L364I, M365N, T370I, D376P, Q377K, A389I, V404A,L406Y, S433K, or G487R.

In some embodiments, the variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 or SEQ ID NO:5 and comprises an amino acid substitution relativeto SEQ ID NO:2 at one or more positions selected from N134, E138, P188,Q377, P405, Q418, S458, L502, R508, K509, and A511, wherein the positionis numbered with reference to SEQ ID NO:2. In some embodiments, thevariant comprises one or more amino acid substitutions selected fromN134S, E138Q, P188S, Q377K, P405V, Q418V, S458Q, L502S, R508D, K509D,and A511T. In some embodiments, the variant has the amino acid sequenceof SEQ ID NO:8, the sequence of Variant 436 described in Table 2.

In some embodiments, the variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 or SEQ ID NO:5 and comprises an amino acid substitution relativeto SEQ ID NO:2 at one or more positions selected from N134, E138, P188,P405, Q418, 5433, S458, L502, R508, K509, and A511, wherein the positionis numbered with reference to SEQ ID NO:2. In some embodiments, thevariant comprises an amino acid substitution at one or more positionsselected from N134S, E138Q, P188S, P405V, Q418V, S433K, S458Q, L502S,R508D, K509D, and A511T. In some embodiments, the variant has the aminoacid sequence of SEQ ID NO:9, the sequence of Variant 438 described inTable 2. In some embodiments, the variant comprises at least about 70%(or at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or at least about 99%) sequence identityto the amino acid sequence of SEQ ID NO:9 and further comprises one ormore amino acid substitutions selected from G14R/V, Q18I, A63R, R65G,V104I/M, N128H, S134R, N174C, N177T, K224R, L226M, E227R, S244P,S283F/M, S306W, G351C, L364I, M365N, T370I, D376P, Q377K, A389I, V404A,L406Y, S433K, or G487R. In some embodiments, the variant comprises theamino acid sequence of SEQ ID NO:9 and further comprises one or moreamino acid substitutions selected from G14R/V, Q18I, A63R, R65G,V104I/M, N128H, S134R, N174C, N177T, K224R, L226M, E227R, S244P,S283F/M, S306W, G351C, L364I, M365N, T370I, D376P, Q377K, A389I, V404A,L406Y, S433K, or G487R.

In some embodiments, the variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 or SEQ ID NO:5 and comprises an amino acid substitution relativeto SEQ ID NO:2 at one or more positions selected from N128, N134, E138,N174, P188, L226, G351, P405, Q418, S433, S458, L502, 8508, K509, andA511, wherein the position is numbered with reference to SEQ ID NO:2. Insome embodiments, the variant comprises one or more amino acidsubstitutions selected from N128H, N134S, E138Q, N174C, P188S, L226M,G351C, P405V, Q418V, S433K, S458Q, L502S, R508D, K509D, and A511T. Insome embodiments, the variant has the amino acid sequence of SEQ IDNO:10, the sequence of Variant 547 described in Table 2.

In some embodiments, the variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 or SEQ ID NO:5 and comprises an amino acid substitution relativeto SEQ ID NO:2 at one or more positions selected from Q18, A63, R65,N128, N134, E138, P188, P405, Q418, S433, S458, G487, L502, R508, K509,and A511, wherein the position is numbered with reference to SEQ IDNO:2. In some embodiments, the variant comprises one or more amino acidsubstitutions selected from Q18I, A63R, R65G, N128H, N134S, E138Q,P188S, P405V, Q418V, S433K, S458Q, G487R, L502S, R508D, K509D, and A511T. In some embodiments, the variant has the amino acid sequence of SEQID NO:11, the sequence of Variant 555 described in Table 2.

In some embodiments, the variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 or SEQ ID NO:5 and comprises an amino acid substitution relativeto SEQ ID NO:2 at one or more positions selected from Q18, N128, N134,E138, N174, N177, P188, L226, G351, P405, Q418, S433, S458, L502, 8508,K509, and A511, wherein the position is numbered with reference to SEQID NO:2. In some embodiments, the variant comprises one or more aminoacid substitutions selected from Q18I, N128H, N134S, E138Q, N174C,N177T, P188S, L226M, G351C, P405V, Q418V, S433K, S458Q, L502S, R508D,K509D, and A511T. In some embodiments, the variant has the amino acidsequence of SEQ ID NO:12, the sequence of Variant 556 described in Table2.

In some embodiments, the variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 or SEQ ID NO:5 and has one or more amino acid substitution setsselected from the amino acid substitution sets listed in Table 2 orTable 5.

In some embodiments, a FAR variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:5 and comprises an amino acid substitution relative to SEQ ID NO:5at one or more positions selected from A2, Q4, Q5, H8, A9, A45, P63,R66, E72, A74, S77, E88, A108, G111, G113, D116, N135, D141, Q181, D199,E205, E238, A374, A375, P406, D411, R412, D422, D430, S434, 1438, N459,E497, Y501, S502, L503, T505, Q508, R509, K510, K511, A512, and A513,wherein the position is numbered with reference to SEQ ID NO:5. In someembodiments, the variant comprises one or more amino acid substitutionsselected from A2F/G/H/P/Q/T, Q4I, Q5F/N, H8K/N, A9L, A45V, P63Q/S, R66N,E72Q/S, A74L, S77G, E88Q, A108C/L/R, G111S, G113A, D116A/E, N135K,D141C/G, Q181D, D199G, E205G/R, E238C, A374V, A375Q/Y, P406S, D411R,R412H, D422A, D430K, S434F/K/W, I438V, N459G/Q, E497F/Y, Y501G/P/S/W,S502G, L503Q/R/S, T505K/R, Q508G/S, R509A/D, K510G, K511C/D/G,A512G/K/P/Q/S/T, and A513L/Y.

In some embodiments, a variant as described herein is encoded by apolynucleotide that hybridizes at high stringency to the complement ofSEQ ID NOs:1, 3, 4, 13, or 14 and comprises one or more amino acidsubstitutions as described herein.

In some embodiments, the invention relates to improved FAR polypeptidescomprising an amino acid sequence that is at least 85% identical to SEQID NO:2 and includes at least one substitution of an amino acid residuecompared to a wild type FAR having at least 85% sequence identity to SEQID NO:2, wherein the improved FAR is capable of producing 1.5 times morefatty alcohol than the corresponding wild-type when assayed under thesame conditions.

In some embodiments, the improved FAR polypeptide comprises an aminoacid sequence that is at least 80% (also at least 85%, also at least 90%and also at least 95%) identical to SEQ ID NO:2 and includessubstitutions at one or more of positions 2, 134, 138, 188, 405 and 511relative to SEQ ID NO: 2. In some embodiments, the substitution of theamino acid residue at position 2 is H, T, D, F, V, G, Q, P or I; thesubstitution of the amino acid residue at position 134 is R, K, or S;the substitution of the amino acid residue at position 138 is Q or L;the substitution of the amino acid residue at position 188 is S; thesubstitution of the amino acid residue at position 405 is V, S, F, G, C,L, S, A, or W; and the substitution of the amino acid residue atposition 511 is T, P, G, S, K, Q, or R. In other embodiments, theimproved FAR includes substitutions at each of positions 2, 134, 138,188, 405 and 511 relative to SEQ ID NO:2. In some embodiments theimproved FAR includes substitutions at the combination of positions 134,138, 188 and 511 relative to SEQ ID NO:2.

In some embodiments, the improved FAR is a functional fragment of animproved full-length FAR enzyme.

In another aspect, the invention relates to an isolated polynucleotidecomprising a sequence encoding an improved FAR polypeptide encompassedby the invention. In some embodiments, the polynucleotide is a codonoptimized polynucleotide.

In another aspect, the invention relates to a vector which comprisessaid polynucleotides and optionally one or more control sequences suchas a promoter capable of mediating expression of the polynucleotideencoding the improved FAR polypeptides in a host microorganism.

In another aspect, the invention relates to a recombinant microorganismcomprising the vector and/or polynucleotide encoding an improved FARpolypeptide encompassed by the invention. In some embodiments, themicroorganism is an E. coli or a yeast (e.g., a Yarrowia or aSaccharomyces cerevisiae).

In yet another aspect, the invention relates to a host cell comprising arecombinant polynucleotide sequence encoding a fatty alcohol formingacyl-CoA reductase (FAR) variant. In some embodiments, the host cellcomprises a FAR variant that has at least 70% (or at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99%) sequence identity to SEQ ID NO:2, whereinthe variant comprises a substitution at one or more positions selectedfrom position 134, position 138, position 511, position 510, position 2,position 140, position 421, and position 458, wherein the position isnumbered with reference to SEQ ID NO:2, with the proviso that the FARvariant does not have the sequence of SEQ ID NO:5. In some embodiments,the FAR variant comprises a substitution at one or more of positions134, 138, and 511, wherein the position is numbered with reference toSEQ ID NO:2, and wherein the amino acid at position 134 is lysine,arginine, or serine; the amino acid at position 138 is leucine orglutamine; and/or the amino acid at position 511 is glycine, isoleucine,lysine, proline, glutamine, arginine, serine, or threonine. In someembodiments, the FAR variant further comprises a substitution at one orboth of positions 510 and 2, wherein the position is numbered withreference to SEQ ID NO:2, and wherein the amino acid at position 510 isalanine, aspartic acid, glycine, proline, serine, or tyrosine; and/orthe amino acid at position 2 is aspartic acid, phenylalanine, glycine,histidine, isoleucine, asparagine, proline, glutamine, threonine,valine, or tryptophan. In some embodiments, the FAR variant furthercomprises a substitution at one or more of positions 140, 421, and 458,wherein the position is numbered with reference to SEQ ID NO:2, andwherein the amino acid at position 140 is cysteine; the amino acid atposition 421 is isoleucine, leucine, asparagine, proline, arginine,serine, valine, or tyrosine; and/or the amino acid at position 458 isglycine, leucine, methionine, or glutamine. In some embodiments, the FARvariant further comprises a substitution at one or more of positions188, 405, and 418, wherein the position is numbered with reference toSEQ ID NO:2, and wherein the amino acid at position 188 is alanine,isoleucine, methionine, or serine; the amino acid at position 405 isalanine, cysteine, phenylalanine, glycine, leucine, serine, valine, ortryptophan; and/or the amino acid at position 418 is isoleucine,arginine, valine, or tyrosine.

In some embodiments, the host cell comprises a FAR variant comprising atleast 95% sequence identity to SEQ ID NO:5. In some embodiments, thehost cell comprises a FAR variant comprising at least 95% sequenceidentity to SEQ ID NO:5 and comprises a substitution at position 135,position 139, and position 512, numbered with reference to SEQ ID NO:5.

In some embodiments, the host cell comprises a FAR variant that has atleast 70% (or at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%)sequence identity to SEQ ID NO:2, wherein the variant comprises asubstitution at one or more positions selected from position 14,position 18, position 63, position 65, position 104, position 128,position 134, position 174, position 177, position 224, position 226,position 227, position 244, position 283, position 306, position 351,position 364, position 365, position 370, position 376, position 377,position 389, position 404, position 406, position 433, or position 487,with the proviso that the FAR variant does not have the sequence of SEQID NO:5. In some embodiments, the substitution or substitutions areselected from G14R/V, Q18I, A63R, R65G, V104I/M, N128H, S134R, N174C,N177T, K224R, L226M, E227R, S244P, S283F/M, S306W, G351C, L364I, M365N,T370I, D376P, Q377K, A389I, V404A, L406Y, S433K, or G487R.

In some embodiments, the host cell comprises a FAR variant that has theamino acid sequence of any of SEQ ID NOs:6, 7, 8, 9, 10, 11, or 12. Insome embodiments, the host cell comprises a FAR variant that comprisesthe amino acid sequence of any of SEQ ID NOs:6, 7, 8, 9, 10, 11, or 12and further comprises a substitution at one or more positions selectedfrom position 14, position 18, position 63, position 65, position 104,position 128, position 134, position 174, position 177, position 224,position 226, position 227, position 244, position 283, position 306,position 351, position 364, position 365, position 370, position 376,position 377, position 389, position 404, position 406, position 433,and position 487. In some embodiments, the substitution or substitutionsare selected from G14R/V, Q18I, A63R, R65G, V104I/M, N128H, S134R,N174C, N177T, K224R, L226M, E227R, S244P, S283F/M, S306W, G351C, L364I,M365N, T370I, D376P, Q377K, A389I, V404A, L406Y, S433K, or G487R.

In one aspect, a FAR variant protein, vectors and cells comprising anucleic acid encoding the FAR variant protein, cells expressing the FARvariant protein, and fatty alcohol products obtained from the cells areprovided, where the FAR variant protein has 100% identity to SEQ: IDNO:2 except for the substitutions present in any individual FAR variantselected from variant numbers 1-629 in Table 2. In other embodiments,the FAR variant protein has 100% identity to SEQ: ID NO:2 except for (1)the substitutions present in any individual FAR variant selected fromvariant numbers 1-629 in Table 2 and (2) one, two, three, four, five,six, seven, eight, nine, ten, eleven or twelve additional substitutions,e.g., 1-5, 2-6, 4-8, 5-12 substitutions (which optionally areconservative substitutions).

In one aspect, a FAR variant protein, vectors and cells comprising anucleic acid encoding the FAR variant protein, cells expressing the FARvariant protein, and fatty alcohol products obtained from the cells areprovided, where the FAR variant protein has 100% identity to SEQ: IDNO:5 except for the substitutions present in any individual FAR variantselected from variant numbers 1-629 in Table 5. In other embodiments,the FAR variant protein has 100% identity to SEQ: ID NO:5 except for (1)the substitutions present in any individual FAR variant selected fromvariant numbers 1-629 in Table 5 and (2) one, two, three, four, five,six, seven, eight, nine, ten, eleven or twelve additional substitutions,e.g., 1-5, 2-6, 4-8, 5-12 substitutions (which optionally areconservative substitutions).

In some embodiments, the host cell is a yeast or a bacterium. In someembodiments, the host cell is E. coli, a Yarrowia species, or aSaccharomyces species.

In some embodiments, the host cell produces more fatty alcohol than acorresponding cell of the same type expressing a wild-type FAR fromwhich the FAR variant is derived. In some embodiments, the host cellproduces at least 1.5, at least 2, at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, or at least 10 timesmore fatty alcohol than the corresponding cell of the same type whichexpresses the wild-type FAR from which the FAR variant is derived. Insome embodiments, at least 90% of the fatty alcohol produced is C10-C18.In some embodiments, at least 90% of the fatty alcohol produced isC12-C16 fatty alcohols. In some embodiments, at least 30% of the fattyalcohol produced is C12-C14 fatty alcohols. In some embodiments, atleast 55% of the fatty alcohol produced is C16-C18 fatty alcohols. Insome embodiments, at least 90% of the fatty alcohol produced is C14-C18fatty alcohols. In some embodiments, the host cell produces a fattyalcohol profile comprising an increased amount of C16:1 (cisΔ⁹-1-hexadecenol) fatty alcohol and a decreased amount of C18:1 (cisΔ¹¹-1-octadecenol) fatty alcohol relative to a corresponding cell of thesame type expressing a wild-type FAR from which the FAR variant isderived. In some embodiments, at least 5 g/L of recoverable fattyalcohols are produced. In some embodiments, at least 15 g/L ofrecoverable fatty alcohols are produced.

In still another aspect, the invention relates to method of producingfatty alcohols comprising culturing a host cell as described herein in aculture medium under conditions in which the fatty alcohols areproduced. In some embodiments, the fatty alcohols are secreted into theculture medium. In some embodiments, the fatty alcohols are recovered(e.g., from the host cell or from the culture medium). In someembodiments, the method further comprises the step of isolating thefatty alcohols from the culture medium. In some embodiments, at least 5g/L of recoverable fatty alcohols are produced. In some embodiments, atleast 15 g/L of recoverable fatty alcohols are produced.

In still another aspect, the invention relates to methods of producingfatty alcohol compositions comprising culturing a microorganism (e.g.,E. coli) comprising a polynucleotide encoding an improved FARpolypeptide according to the invention in a suitable culture medium,allowing expression of the polynucleotide and production of the fattyalcohols. In some embodiments at least 5.0 g/L (e.g., at least 10.0 g/L,at least 15 g/L, at least 20 g/L, at least 25 g/L, at least 30 g/L, atleast 35 g/L, at least 40 g/L, at least 45 g/L, or at least 50 g/L) offatty alcohols are recovered from the microorganism and/or culture. Insome embodiments the fatty alcohols produced by the recombinantmicroorganism are further isolated from the culture.

In yet another aspect, the invention relates to compositions comprisingand/or derived from the fatty alcohol composition produced by themethods of the invention. In some embodiments, the fatty alcoholcompositions produced by the methods encompassed by the invention, or afraction thereof, are further reduced to yield an alkane composition. Insome embodiments the fatty alcohol composition produced by the methodsencompassed by the invention are esterified yielding fatty esters. Insome embodiments, the fatty alcohol compositions produced by the methodsencompassed by the invention, or a fraction thereof, are modified toproduce fatty esters. In some embodiments, the fatty alcohols aresubjected to reduction or esterification to produce a diesel fuelcomponent.

In yet another aspect, the invention provides methods of producing adetergent composition, the method comprising combining the fattyalcohols produced by the methods described herein, or a fractionthereof, with a detergent component selected from sodium carbonate, acomplexation agent, zeolites, a protease, a lipase, amylase,carboxymethyl cellulose, optical brighteners, colorants and perfumes,thereby producing the detergent composition. In another aspect, theinvention relates to detergent compositions produced by a methoddescribed herein.

In still another aspect, the invention provides methods of producing afuel composition, the method comprising reducing or esterifying thefatty alcohols produced by a method described herein, or a fractionthereof, to yield the fuel composition. In some embodiments, a method ofproducing fuel comprises (a) producing fatty alcohols according to amethod described herein, and (b) subjecting the fatty alcohols, or afraction thereof, to one or more chemical reactions to generate alkanes,whereby fuel is produced. In some embodiments, the fatty alcoholscomprise at least 90% C14-C18 fatty alcohols. In some embodiments, thefatty alcohols comprise less than 1% C18:0 fatty alcohols. In anotheraspect, the invention relates to fuel compositions produced by a methoddescribed herein.

In a first embodiment, the present invention relates to an improvedfatty alcohol forming acyl-CoA reductase (FAR) polypeptide capable ofproducing at least about 1.5 times more fatty alcohol than a wild-typeFAR polypeptide comprising SEQ ID NO:2 when assayed under the sameconditions.

In a second embodiment, the present invention relates to an improvedfatty alcohol forming acyl-CoA reductase (FAR) polypeptide comprising anamino acid sequence that is at least 80% identical to SEQ ID NO:2 andincludes at least one substitution of an amino acid residue compared towild-type FAR having at least 80% sequence identity to SEQ ID NO:2,wherein the improved FAR is capable of producing 1.5 times more fattyalcohol than the corresponding wild-type when assayed under the sameconditions.

In a third embodiment, the present invention relates to the FARpolypeptide of the first or second embodiments, which comprises an aminoacid sequence that is at least 90% identical to SEQ ID NO:2.

In a fourth embodiment, the present invention relates to the FARpolypeptide of the first or second embodiments, which comprises an aminoacid sequence that is at least 90% identical to SEQ ID NO:2 and includessubstitutions at one or more of positions 2, 134, 138, 188, 405 and 511relative to SEQ ID NO:2.

In a fifth embodiment, the present invention relates to the FARpolypeptide of the first or second embodiments, which comprises an aminoacid sequence that is at least 95% identical to SEQ ID NO:2 and includessubstitutions at one or more of positions 2, 134, 138, 188, 405 and 511relative to SEQ ID NO:2.

In a sixth embodiment, the present invention relates to the FARpolypeptide of the fourth embodiment in which the amino acid residue atposition 2 is H, T, D, F, V, G, Q, P or I; the amino acid residue atposition 134 is R, K, or S; the amino acid residue at position 138 is Qor L; the amino acid residue at position 188 is S; the amino acidresidue at position 405 is V, S, F, G, C, L, S, A, or W; and the aminoacid residue at position 511 is T, P, G, S, K, Q, or R.

In a seventh embodiment, the present invention relates to the FARpolypeptide of the fourth embodiment which includes substitutions ateach of positions 2, 134, 138, 188, 405, and 511 relative to SEQ IDNO:2.

In an eighth embodiment, the present invention relates to the FARpolypeptide of the fourth embodiment which includes substitutions ateach of positions 134, 138, 188, and 511 relative to SEQ ID NO:2.

In a ninth embodiment, the present invention relates to the FARpolypeptide of the eighth embodiment in which the substitution atposition 134 is S; the substitution at position 138 is Q; thesubstitution at position 199 is S; and the substitution at position 511is T.

In a tenth embodiment, the present invention relates to the FARpolypeptide of the fourth embodiment which comprises one to fiveadditional substitutions at positions other than positions 2, 134, 138,188, 405, and 511 relative to SEQ ID NO:2.

In an eleventh embodiment, the present invention relates to the FARpolypeptide of the tenth embodiment in which the additionalsubstitutions are selected from the substitutions in Table 2.

In a twelfth embodiment, the present invention relates to the FARpolypeptide of the first or second embodiments which is a polypeptideselected from the variant polypeptides in Table 2.

In a thirteenth embodiment, the present invention relates to the FARpolypeptide of any of the first through twelfth embodiments which is afunctional fragment.

In a fourteenth embodiment, the present invention relates to an isolatedpolynucleotide comprising a sequence encoding a FAR polypeptideaccording to any one of the first through thirteenth embodiments.

In a fifteenth embodiment, the present invention relates to anexpression vector comprising a nucleic acid sequence encoding a FARpolypeptide according to any one of the first through thirteenthembodiments operably linked to a control sequence suitable for effectingexpression in a host cell.

In a sixteenth embodiment, the present invention relates to theexpression vector of the fifteenth embodiment in which the controlsequence is a promoter.

In a seventeenth embodiment, the present invention relates to theexpression vector of the sixteenth embodiment which is suitable foreffecting expression in a bacterium, yeast, algae or filamentous fungi.

In an eighteenth embodiment, the present invention relates to theexpression vector of the seventeenth embodiment which is suitable foreffecting expression in an E. coli.

In a nineteenth embodiment, the present invention relates to theexpression vector of the seventeenth embodiment which is suitable foreffecting expression in Saccharomyces cerevisiae.

In a twentieth embodiment, the present invention relates to theexpression vector of the sixteenth embodiment which is suitable foreffecting expression in a Yarrowia sp.

In a twenty-first embodiment, the present invention relates to amicroorganism engineered to express a FAR polypeptide according to anyone of the first through thirteenth embodiments.

In a twenty-second embodiment, the present invention relates to themicroorganism of the twenty-first embodiment which is an E. coli.

In a twenty-third embodiment, the present invention relates to themicroorganism of the twenty-first embodiment which is a Yarrowia.

In a twenty-fourth embodiment, the present invention relates to themicroorganism of the twenty-first embodiment which is a Saccharomycescerevisiae.

In a twenty-fifth embodiment, the present invention relates to a methodof producing a fatty alcohol composition comprising culturing amicroorganism according to any one of the twenty-first throughtwenty-fourth embodiments in a suitable culture medium, in which thefatty alcohol composition is produced.

In a twenty-sixth embodiment, the present invention relates to themethod of the twenty-fifth embodiment, in which at least 5 g/L ofrecoverable fatty alcohols are produced.

In a twenty-seventh embodiment, the present invention relates to themethod of the twenty-sixth embodiment, in which at least 15 g/L ofrecoverable fatty alcohols are produced.

In a twenty-eighth embodiment, the present invention relates to themethod according to the twenty-fifth through twenty-seventh embodiments,which further comprises the step of isolating the fatty alcohols fromthe culture.

In a twenty-ninth embodiment, the present invention relates to the fattyalcohol composition produced by the method of the twenty-fifth throughtwenty-eighth embodiments.

In a thirtieth embodiment, the present invention relates to a method ofproducing an alkane composition comprising culturing a microorganismaccording to any one of the twenty-first through twenty-fourthembodiments in a suitable culture medium in which a fatty alcoholcomposition is produced and reducing the fatty alcohol composition toyield an alkane composition.

In a thirty-first embodiment, the present invention relates to thealkane composition produced by the method of the thirtieth embodiment.

In a thirty-second embodiment, the present invention relates to a methodof producing a fatty ester composition comprising culturing amicroorganism according to any one of the twenty-first throughtwenty-fourth embodiments in a suitable culture medium in which a fattyalcohol composition is produced, exposing the fatty alcohol compositionto esterification to produce a fatty ester composition.

In a thirty-third embodiment, the present invention relates to the fattyalcohol composition produced by the method of the thirty-secondembodiment.

These and other embodiments of the invention are more fully describedherein below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts biosynthetic pathways for fatty alcohol production via(a) an acyl-CoA dependent pathway utilizing fatty acyl-ACP and fattyacyl-CoA intermediates and (b) an acyl-CoA independent pathway utilizingfatty acyl-ACP intermediates but not a fatty acyl-CoA intermediate,wherein “R” as used in the compound formulas is a C7 to C23 saturated,unsaturated, linear, branched or cyclic hydrocarbon chain.

FIG. 2 depicts a sequence alignment of the wild-type FAR enzyme ofMarinobacter algicola strain DG893 (SEQ ID NO:2) and the wild-type FARenzyme of Marinobacter aquaeolei (SEQ ID NO:5).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains. Generally,the nomenclature used herein and the laboratory procedures of cellculture, molecular genetics, organic chemistry, analytical chemistry andnucleic acid chemistry described below are those well known and commonlyemployed in the art. It is noted that the indefinite articles “a” and“an” and the definite article “the” are used in the present applicationto mean one or more unless the context clearly dictates otherwise.Further, the term “or” is used in the present application to mean thedisjunctive “or” and the conjunctive “and”.

Abbreviations: “FAR” denotes fatty acyl reductase or fatty alcoholforming acyl-CoA reductase; “ACP” denotes acyl carrier protein; “CoA”denotes coenzyme A; “TE” denotes thioesterase; “FAS” denotes fatty acidsynthase; “FACR” denotes fatty acyl-CoA reductase; “FACS” denotes fattyacyl-CoA synthase (synthetase) and acyl-CoA synthase (synthetase) asused interchangeably herein; and “ACC” denotes acetyl-CoA carboxylase.Amino acids are designated using the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission.

“Fatty alcohol forming acyl-CoA reductase,” “fatty acyl reductase” and“FAR”, are used interchangeably herein to refer to an enzyme thatcatalyzes the reduction of a fatty acyl-CoA, a fatty acyl-ACP, or otherfatty acyl thioester complex to a fatty alcohol, in a reaction linked tothe oxidation of NAD(P)H to NAD(P)⁺, as shown in the following Scheme 1:

wherein “R” represents a C7 to C23 saturated, unsaturated, linear,branched or cyclic hydrocarbon chain, and “R₁” represents CoA, ACP orother fatty acyl thioester substrates. CoA is a non-protein acyl carriergroup factor (or moiety) involved in the synthesis and oxidation offatty acids. “ACP” is a polypeptide or protein subunit of fatty acidsynthase used in the synthesis of fatty acids. In some embodiments, afatty aldehyde intermediate may be produced in the reaction depicted inScheme 1.

FARs are distinct from FACRs. FACRs reduce fatty acyl-CoA intermediatesonly to fatty aldehydes and require an additional oxidoreductase enzymeto generate the corresponding fatty alcohol.

“Fatty aldehyde” as used herein refers to a saturated or unsaturatedaliphatic aldehyde and reference is made to FIG. 1, wherein R is asdefined above.

The term “fatty acid” as used herein refers to a compound of formulaIII:

wherein “R” is as defined above. Saturated or unsaturated fatty acidscan be described as “Ca:b”, wherein “a” is an integer that representsthe total number of carbon atoms and “b” is an integer that refers tothe number of double bonds in the carbon chain.

The term “fatty alcohol” as used herein refers to an aliphatic alcoholof the formula R—OH, where R is as defined above. Saturated orunsaturated fatty alcohols can also be described using the nomenclature“Ca:b” or, alternatively “Ca:b-OH”, wherein “a” is an integer thatrepresents the total number of carbon atoms in the fatty alcohol and “b”is an integer that refers to the number of double bonds in the carbonchain. In some embodiments, a fatty alcohol produced according to themethods disclosed herein is a C8-C24 saturated or unsaturated fattyalcohol (i.e., a C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18,C19, C20, C21, C22, or C24 fatty alcohol). In some embodiments, one ormore of the following fatty alcohols is produced: 1-octanol (C8:0),1-decanol (C10:0), 1-dodecanol (012:0), 1-tetradecanol (C14:0),1-hexadecanol (C16:0), 1-octadecanol (C18:0), 1-icosanol (C20:0),1-docosanol (C22:0), 1-tetracosanol (024:0), cis Δ⁹-1-hexadecenol(C16:1), and cis Δ¹¹-1-octadecenol (C18:1). It is understood that,unless otherwise specified, a reference to a “Cx fatty alcohol” includesboth saturated and unsaturated fatty alcohols having “x” carbon atoms.

Unsaturated fatty acids or fatty alcohols can be referred to as “cisΔ^(x)” or “trans Δ^(z)”, wherein “cis” and “trans” refer to the carbonchain configuration around the double bond and “x” indicates the numberof the first carbon of the double bond, wherein carbon 1 is thecarboxylic acid carbon of the fatty acid or the carbon bound to the —OHgroup of the fatty alcohol.

The terms “fatty acyl-thioester” and “fatty acyl-thioester complex”refer to a compound of formula (I), in which a fatty acyl moiety iscovalently linked via a thioester linkage to a carrier moiety. Fattyacyl-thioesters are substrates for the improved FAR polypeptidesdescribed herein.

The term “fatty acyl-CoA” refers to a compound of formula (I), whereinR₁ is Coenzyme A.

The term “fatty acyl-ACP” refers to a compound of formula (I) wherein R₁is acyl carrier protein.

The term “fatty acid synthase (FAS)” refers to an enzyme or enzymecomplex that catalyzes the conversion of acetyl-CoA and malonyl-CoA tofatty acyl-ACP as set forth in the following Scheme 2:

wherein ACP is a protein which comprises a covalently attachedphosphopantetheine moiety. In certain embodiments, the FAS is composedof more than one distinct enzymatic activity. In various embodiments,the distinct enzymatic activities reside in separate polypeptides. Insome embodiments, the separate polypeptides form one or more proteincomplexes.

The term “acyl-ACP thioesterase (TE)” refers to an enzyme that catalyzesthe cleavage of acyl-ACP to form a fatty acid, as shown in the followingScheme 3, wherein R has the same meaning as set forth above:

The term “fatty acyl-CoA synthetase” or “acyl-CoA synthetase” or “FACS”are used interchangeably herein to refer to an enzyme that catalyzes theformation of a covalent complex between the acyl portion of the fattyacid and CoA as shown in the following Scheme 4, wherein R has the samemeaning as set forth above:

The term “acetyl-CoA carboxylase (ACC)” refers to an enzyme thatcatalyzes the conversion of acetyl-CoA to malonyl-CoA as shown in thefollowing Scheme 5:

The term “acyl-CoA dehydrogenase (ACD)” refers an enzyme that catalyzesthe introduction of a trans double-bond between C2 and C3 of an acyl-CoAthioester substrate as shown in the following Scheme 6:

The phrase “acyl-CoA independent pathway” refers to the production offatty alcohols by the direct enzymatic conversion of fatty acyl-ACPsubstrates to fatty alcohols and does not involve the use of free fattyacids or fatty acyl-CoA intermediates. This biosynthetic pathway differsfrom a) the fatty acyl-CoA dependent pathway which converts fattyacyl-ACP directly to fatty acyl CoA via an acyl-transfer reaction, suchas in yeast, and b) the fatty acyl-CoA dependent pathway which convertsfatty acyl-ACP to fatty acyl-CoA via a free fatty acid intermediate,such as in bacteria. See FIG. 1.

The acyl-CoA independent pathway has the advantage of bypassing the stepof forming a fatty acyl-CoA substrate from free fatty acid, whichrequires the use of ATP. Therefore, the acyl-CoA independent pathway mayuse less energy than the acyl-CoA dependent pathway that utilizes a freefatty acid intermediate.

“Conversion” refers to the enzymatic conversion of the substrate to thecorresponding product.

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation. A wild-type organism or cell refers toan organism or cell that has not been intentionally modified by humanmanipulation.

The term “wild-type fatty alcohol forming acyl-CoA reductase” or“wild-type FAR,” as used herein, refers to a naturally-occurring FARpolypeptide that is produced in nature. In some embodiments, a wild-typeFAR is produced by a gammaproteobacteria, including but not limited tostrains of Marinobacter, Oceanobacter, and Hahella. Naturally occurringFAR polypeptides are described, for example, in US patent publication2011/0000125, incorporated by reference herein. In some embodiments, awild-type FAR is a naturally-occurring FAR polypeptide that is producedby the Marinobacter algicola strain DG893 (SEQ ID NO:2). In someembodiments, a wild-type FAR is a naturally-occurring FAR polypeptidethat is produced by the Marinobacter aquaeolei strain VT8 (SEQ IDNO:5)′. FARs that are not wild-type can be denoted “recombinant” FARs,whether prepared using recombinant techniques or by chemical synthesis.

The terms “improved FAR polypeptides” and “FAR variants” are usedinterchangeably to refer to improved full length FAR enzymes havingsubstitutions at one or more positions relative to a wild type FARenzyme. and functional (or “biologically active”) fragments thereof. Inone embodiment, “improved FAR polypeptides” comprise at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to SEQ IDNO:2 and also the functional fragments of said improved full length FARenzymes. “Improved FAR polypeptides” can also refer to polypeptidescomprising at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 5, and functional fragments thereof.

“Variant” means an improved FAR polypeptide or polynucleotide encodingan improved FAR polypeptide comprising one or more modificationsrelative to a wild-type FAR polypeptide such as wild-type FAR fromMarinobacter species, or the wild-type polynucleotide such assubstitutions, insertions, and/or deletions of one or more amino acidresidues or of one or more specific nucleotides or codons in thepolypeptide or polynucleotide.

The terms “modifications” and “mutations,” when used in the context ofsubstitutions, deletions, insertions and the like with respect topolynucleotides and polypeptides, are used interchangeably herein andrefer to changes that are introduced by genetic manipulation to createvariants from a wild-type sequence.

“Deletion” refers to modification to a polypeptide by removal of one ormore amino acids from the reference polypeptide. Deletions can compriseremoval of 1 or more amino acids, 2 or more amino acids, 5 or more aminoacids, 10 or more amino acids, 15 or more amino acids, or 20 or moreamino acids, up to 10% of the total number of amino acids, or up to 20%of the total number of amino acids making up the reference enzyme whileretaining enzymatic activity and/or retaining the improved properties ofan engineered enzyme. Deletions can be directed to the internal portionsand/or terminal portions of the polypeptide. In various embodiments, thedeletion can comprise a continuous segment or can be discontinuous. Theterm “deletion” is also used to refer to a DNA modification in which oneor more nucleotides or nucleotide base-pairs have been removed, ascompared to the corresponding reference, parental or “wild type” DNA.

“Insertion” refers to modification to a polypeptide by addition of oneor more amino acids to the reference polypeptide. In some embodiments,the modification comprises insertions of one or more amino acids to thenaturally occurring polypeptide as well as insertions of one or moreamino acids to other modified polypeptides. Insertions can be in theinternal portions of the polypeptide, or to the carboxy or aminoterminus. Insertions as used herein include fusion proteins as is knownin the art. The insertion can be a contiguous segment of amino acids orseparated by one or more of the amino acids in the naturally occurringpolypeptide. The term “insertion” is also used to refer to a DNAmodification in which or more nucleotides or nucleotide base-pairs havebeen inserted, as compared to the corresponding reference, parental or“wild type” DNA.

“Different from” or “differs from” with respect to a designatedreference sequence refers to difference of a given amino acid orpolynucleotide sequence when aligned to the reference sequence.Generally, the differences can be determined when the two sequences areoptimally aligned. Differences include modifications such as insertions,deletions, or substitutions of amino acid residues in comparison to thereference sequence.

A polynucleotide or polypeptide that is “derived from” a particularorganism refers to a wild-type polynucleotide or polypeptide thatoriginates in the organism. The expression “derived from” can also beused in the context of mutant or variant polynucleotides andpolypeptides. In this context, the particular mutant is said to be“derived from” the wild-type sequence from which it was engineered or towhich its sequence is compared. For example, in some embodiments, animproved FAR enzymes described herein can be said to be “derived from”the wild-type FAR enzyme of SEQ ID NO:2 or SEQ ID NO:5. In someembodiments a polypeptide sequence of an engineered FAR is at least 90%,sometimes at least 95%, sometimes at least 96%, sometimes at least 97%,and sometimes at least 98% identical to the naturally occurring homologfrom which it was derived. In some embodiments a polypeptide sequence ofan engineered FAR has 100% identity with the naturally occurringhomolog, except at a position or set of positions specified (relative toSEQ ID NO:2) in Table 2, below (i.e., corresponding to one of Variants1-629.

“Percentage of sequence identity,” “percent identity” and “percentagehomology” are used interchangeably herein to refer to comparisons amongpolynucleotides and polypeptides, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which may also contain gaps to optimize thealignment) for alignment of the two sequences. The percentage may becalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparison(including positions where one of the sequences has a gap(s) andmultiplying the result by 100 to yield the percentage of sequenceidentity. For example, a polypeptide with an amino acid sequencematching SEQ ID NO:2 at 491 positions, with one gap, would have491/512=95.9% identity to SEQ ID NO:2. Similarly, a FAR variant that has475 residues (i.e., less than full-length) and matches SEQ ID NO:2 at460 positions would have 460/475=96.8% identity. Those of skill in theart appreciate that there are many established algorithms available toalign two sequences and that different methods may give slightlydifferent results.

Alignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math.2:482, by the homology alignment algorithm of Needleman and Wunsch,1970, J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the GCG Wisconsin Software Package), or by visualinspection (see generally, Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1995Supplement) (Ausubel)). The Clustral (Chenna R., Sugawara H., Koike T.,Lopez R., Gibson T. J., Higgins D. G., Thompson J. D., (2003) Multiplesequence alignment with the Clustral series of programs, Nucleic AcidsRes., 31, 3497-3500.) and T-Coffee (T-COFFEE: A novel method formultiple sequence alignments. Notredame, Higgins, Heringa, JMB 302(205-217) 2000 software packages may also be used to align sequences.

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., 1990, J. Mol. Biol.215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Informationwebsite. This algorithm involves first identifying high scoring sequencepairs (HSPs) by identifying short words of length W in the querysequence, which either match or satisfy some positive-valued thresholdscore T when aligned with a word of the same length in a databasesequence. T is referred to as, the neighborhood word score threshold(Altschul et al, supra). These initial neighborhood word hits act asseeds for initiating searches to find longer HSPs containing them. Theword hits are then extended in both directions along each sequence foras far as the cumulative alignment score can be increased. Cumulativescores are calculated using, for nucleotide sequences, the parameters M(reward score for a pair of matching residues; always >0) and N (penaltyscore for mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA89:10915). Exemplary determination of sequence alignment and % sequenceidentity can employ the BESTFIT or GAP programs in the GCG WisconsinSoftware package (Accelrys, Madison Wis.), using default parametersprovided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, at least 100 residues in length or the fulllength of the nucleic acid or polypeptide. Since two polynucleotides orpolypeptides may each (1) comprise a sequence (i.e., a portion of thecomplete sequence) that is similar between the two sequences, and (2)may further comprise a sequence that is divergent between the twosequences, sequence comparisons between two (or more) polynucleotides orpolypeptide are typically performed by comparing sequences of the twopolynucleotides over a “comparison window” to identify and compare localregions of sequence similarity.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well-characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. As used herein, the term “stringenthybridization wash conditions” in the context of nucleic acidhybridization experiments, such as Southern and Northern hybridizations,are sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993) “Laboratory Techniques in biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes,” Part I,Chapter 2 (Elsevier, New York), which is incorporated herein byreference. For polynucleotides of at least 100 nucleotides in length,low to very high stringency conditions are defined as follows:prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200μg/ml sheared and denatured salmon sperm DNA, and either 25% formamidefor low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures. For polynucleotides ofat least 100 nucleotides in length, the carrier material is finallywashed three times each for 15 minutes using 2×SSC, 0.2% SDS at least at50° C. (low stringency), at least at 55° C. (medium stringency), atleast at 60° C. (medium-high stringency), at least at 65° C. (highstringency), and at least at 70° C. (very high stringency).

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organism.Although the genetic code is degenerate in that most amino acids arerepresented by several codons, called “synonyms” or “synonymous” codons,it is well known that codon usage by particular organisms is nonrandomand biased towards particular codon triplets. This codon usage bias maybe higher in reference to a given gene, genes of common function orancestral origin, highly expressed proteins versus low copy numberproteins, and the aggregate protein coding regions of an organism'sgenome. In some embodiments, the polynucleotides encoding enzymes may becodon optimized for optimal production from the host organism selectedfor expression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (See GCG Codon Preference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney, J. 0,1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables areavailable for a growing list of organisms (see for example, Wada et al.,1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl.Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASMPress, Washington D.C., p. 2047-2066). The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (ESTs), or predictedcoding regions of genomic sequences (see for example, Mount, D.,Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E.C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput.Appl. Biosci. 13:263-270).

In describing the various variants and amino acid substitutions of thepresent invention, the nomenclature described below is adapted for easeof reference. In all cases the accepted IUPAC single letter or tripleletter amino acid abbreviations are employed. IUPAC single letter aminoacid abbreviations are as follows: alanine (A); cysteine (C); asparticacid (D); glutamic acid (E); phenylalanine (F); glycine (G); histidine(H); isoleucine (I); lysine (K); leucine (L); methionine (M); asparagine(N); proline (P); glutamine (Q); arginine (R); serine (S); threonine(T); valine (V); tryptophan (W); and tyrosine (Y). For amino acidsubstitutions the following nomenclature is used: [Original amino acid,position, substituted amino acid]. As a non-limiting example, for avariant polypeptide described with reference to SEQ ID NO:2, “A2V”indicates that in the variant polypeptide, the alanine at position 2 ofthe reference sequence is replaced by valine, with amino acid positionbeing determined by optimal alignment of the variant sequence with SEQID NO:2. Similarly, “A512K/S/T” describes three variants: a variant inwhich the alanine at position 512 of the reference sequence is replacedby lysine, a variant in which the alanine at position 512 of thereference sequence is replaced by serine, and a variant in which thealanine at position 512 of the reference sequence is replaced bythreonine. In some embodiments, an amino acid (or base) may be called“X,” by which is meant any amino acid (or base). For example,X2D/F/G/H/I/P/N/Q/T/V/W can refer to a substitution in a FAR homolog inwhich the residue (X) at the position in the homolog corresponding toposition 2 of a specified sequence (e.g., SEQ ID NO:2) is substituted sothat the residue at position 2 is any of D, F, G, H, I, P, N, Q, T, V,and W.

The term “amino acid substitution set” or “substitution set” refers to agroup of amino acid substitutions. A substitution set can have 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acidsubstitutions. In some embodiments, a substitution set refers to the setof amino acid substitutions that is present in any of the variant FARpolypeptides listed in Table 2, Table 4, and/or Table 5. For example,the substitution set for Variant 370 (Table 2) consists of the aminoacid substitutions N134S, E138Q, P188S, P405V, Q418V, and A511T.

“Conservative” amino acid substitutions or mutations refer to theinterchangeability of residues having similar side chains, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. However, as used herein, conservative mutations do not includesubstitutions from a hydrophilic to hydrophilic, hydrophobic tohydrophobic, hydroxyl-containing to hydroxyl-containing, or small tosmall residue, if the conservative mutation can instead be asubstitution from an aliphatic to an aliphatic, non-polar to non-polar,polar to polar, acidic to acidic, basic to basic, aromatic to aromatic,or constrained to constrained residue. Further, as used herein, A, V, L,or I can be conservatively mutated to either another aliphatic residueor to another non-polar residue. In some embodiments, conservativelysubstituted variations of the FAR polypeptides of the present inventioninclude substitutions of less than 10%, less than 5%, less than 2% andsometimes less than 1% of the amino acids of the polypeptide sequence,with a conservatively selected amino acid of the same conservativesubstitution group. The addition of sequences which do not alter theencoded activity of an FAR polynucleotide, such as the addition of anon-functional or non-coding sequence, is considered a conservativevariation of the polynucleotide. Table 1 below shows exemplaryconservative substitutions.

TABLE 1 Conservative Substitutions Residue Possible ConservativeMutations A, L, V, I Other aliphatic (A, L, V, I) Other non-polar (A, L,V, I, G, M) G, M Other non-polar (A, L, V, I, G, M) D, E Other acidic(D, E) K, R Other basic (K, R) P, H Other constrained (P, H) N, Q, S, TOther polar (N, Q, S, T) Y, W, F Other aromatic (Y, W, F) C NoneIn some embodiments, there may be at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 15, at least 20, at least 25, at least 30, at least35, or at least 40 conservative substitutions.

“Non-conservative substitution” refers to substitution or mutation of anamino acid in the polypeptide with an amino acid with significantlydiffering side chain properties. Non-conservative substitutions may useamino acids between, rather than within, the defined groups listedabove. In one embodiment, a non-conservative mutation affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain.

“Functional fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion and/or internaldeletion, but where the remaining amino acid sequence is identical tothe corresponding positions in the sequence to which it is beingcompared (e.g., a full-length FAR variant of the invention) and thatretains substantially all of the activity of the full-lengthpolypeptide. Improved functional fragments can comprise up to 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the improvedfull-length FAR enzyme.

An “endogenous” polynucleotide, gene, promoter or polypeptide refers toany polynucleotide, gene, promoter or polypeptide that originates in aparticular host cell. A polynucleotide, gene, promoter or polypeptide isnot endogenous to a host cell if it has been removed from the host cell,subjected to laboratory manipulation, and then reintroduced into a hostcell.

A “heterologous” polynucleotide, gene, promoter or polypeptide refers toany polynucleotide, gene, promoter or polypeptide that is introducedinto a host cell that is not normally present in that cell, and includesany polynucleotide, gene, promoter or polypeptide that is removed fromthe host cell and then reintroduced into the host cell.

“Inactive” or “inactivated” in reference to a gene refers to a genehaving at least one function that is impaired. Genes can be inactivatedin a variety of ways known in the art, including but not limited toinsertion of a mobile genetic element (e.g., a transposon); deletion ofall or part of the gene, such that the gene product is not made, or istruncated and is non-functional; mutation of the gene such that the geneproduct is not made, or is truncated and is non-functional; deletion ormutation of one or more control elements that control expression of thegene such that the gene product is not made; and the like. In certainembodiments genes can be inactivated by methods other than geneticmodification, for example, by gene silencing at the transcriptionallevel or at the post-transcriptional level using for example RNAi.

“Recombinant host cell” refers to a cell into which has been introduceda heterologous polynucleotide, gene, promoter, e.g., an expressionvector, or to a cell having a heterologous polynucleotide or geneintegrated into the genome.

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polypeptide of thepresent disclosure. Each control sequence may be native or foreign tothe nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide.

“Operably linked” and “operably associated” are defined herein as aconfiguration in which a control sequence is appropriately placed at aposition relative to the coding sequence of the DNA sequence such thatthe control sequence directs the expression of a polynucleotide and/orpolypeptide.

“Promoter sequence” is a nucleic acid sequence that is recognized by ahost cell for expression of the coding region. The control sequence maycomprise an appropriate promoter sequence. The promoter sequencecontains transcriptional control sequences, which mediate the expressionof the polypeptide. The promoter may be any nucleic acid sequence whichshows transcriptional activity in the host cell of choice includingmutant, truncated, and hybrid promoters, and may be obtained from genesencoding extracellular or intracellular polypeptides either endogenousor heterologous to the host cell.

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The terms “transform” or “transformation,” as used in reference to acell, means a cell has a non-native nucleic acid sequence integratedinto its genome or as an episome (e.g., plasmid) that is maintainedthrough multiple generations.

The term “culturing” refers to growing a population of microbial cellsunder suitable conditions in a liquid or solid medium. In particularembodiments, culturing refers to the fermentative bioconversion of asubstrate to an end product.

The term “recoverable,” as used in reference to producing a composition(e.g., fatty alcohols) by a method of the present invention, refers tothe amount of composition which can be isolated from the reactionmixture yielding the composition according to methods known in the art.

The term “fuel component,” as used herein, refers to any compound ormixture of compounds that is used to formulate a fuel composition.

II. Introduction

The present invention relates to, among other things, variant FARenzymes and functional fragments thereof with improved properties,polynucleotides encoding the variant FAR enzymes, recombinantmicroorganisms comprising a nucleic acid encoding an improved FARpolypeptide, microorganisms capable of expressing the improved FARpolypeptides, processes for producing fatty alcohols and othercompositions derived therefrom using the improved FAR polypeptides, andthe resultant compositions.

Wild-type FAR polypeptides have been described in WO 2011/008535(published 20 Jan. 2011), incorporated by reference herein for allpurposes. Certain FAR enzymes isolated from genera of the class ofmarine bacteria such as gammaproteobacteria found in seawater (andparticularly FARs obtained from strains of Marinobacter and Oceanobacteror taxonomic equivalents thereof) are capable of generating high yieldsof fatty alcohols when genes encoding these enzymes are expressed inheterologous cells. For example, Marinobacter species algicola (strainDG893) possesses FAR enzymes which are capable of producing more than100-fold more total fatty alcohol than FAR enzymes from B. mori, whenexpressed in an E. coli host. It has now been discovered that FARenzymes containing mutations in their polypeptide sequences, such as oneor more amino acid substitutions, have improved properties as comparedto their wild-type counterparts.

SEQ ID NO:2 is the wild-type FAR from Marinobacter algicola (strainDG893) and is described in WO 2011/008535 (published 20 Jan. 2011),which is incorporated herein by reference for all purposes. SEQ ID NO:13is the polynucleotide sequence encoding SEQ ID NO:2. SEQ ID NO:1 is apolynucleotide sequence encoding the wild-type FAR protein fromMarinobacter algicola strain DG893 (SEQ ID NO: 2) that is codonoptimized for expression in E. coli. SEQ ID NO:3 is a polynucleotidesequence encoding SEQ ID NO:2 that is codon optimized for expression inYarrowia lipolytica. SEQ ID NO:5 is the wild-type FAR from Marinobacteraquaeolei VT8, which is also described in WO 2011/008535. SEQ ID NO:4 isa polynucleotide sequence encoding the wild-type FAR protein fromMarinobacter aquaeolei VT8 (SEQ ID NO:5) that is codon optimized forexpression. Amino acid sequence identity between SEQ ID NO:2 and SEQ IDNO:5 is 78%. See FIG. 2.

In one aspect, the disclosure provides the improved FAR polypeptides perse. In another aspect, it can be seen that substitutions introduced atnumerous different amino acid (also referred to herein as “residue”)positions within the wild-type FAR of SEQ ID NO:2 yield improved FARpolypeptides capable of catalyzing increased production of fattyalcohols as compared to the wild-type FAR (see Table 2). Depending uponthe position mutated, single amino acid changes at specified positionsgive rise to 2-fold or greater increases in fatty alcohol production.

In one aspect the invention relates to FAR polypeptides with at leastabout 70% sequence identity with SEQ ID NO:2 (wild-type M. algicola FAR)and with one or more of the specified substitutions; these polypeptidesexhibit improved characteristics and/or properties as described herein.In some embodiments, a FAR polypeptide shows at least about 75% (e.g.,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% and at least 99%) sequence identity with the wild-type FAR ofSEQ ID NO:2 and also includes one or more of the specified substitutionswill exhibit improved characteristics and/or properties as describedherein. In another aspect, the invention relates to FAR polypeptidesthat comprise an amino acid sequence encoded by a nucleic acid thathybridizes under stringent conditions over substantially the entirelength of a nucleic acid corresponding to SEQ ID NO:1, SEQ ID NO:3, orSEQ ID NO:13. In some embodiments the FAR variant is derived from aMarinobacter species other than Marinobacter algicola.

In a related aspect, the invention describes FAR variants showing atleast about 70% (e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%)sequence identity with SEQ ID NO:5 (wild-type M. aquaeolei FAR) andcomprising substitutions disclosed herein (see, e.g., Tables 4 and 5).These FAR variants exhibit improved characteristics and/or properties asdescribed herein. In some embodiments the FAR variant is derived from aMarinobacter species other than Marinobacter aquaeolei.

III. Improved Properties of Far Variants

In one aspect, the invention provides FAR variants having improvedproperties over a wild-type FAR enzyme (e.g., SEQ ID NO:2 or 5) or overa reference sequence. For example, a cell expressing a FAR variant ofthe invention may have increased fatty alcohol production compared to acell expressing a wild-type FAR (also referred to herein as a “controlcell”), and/or the fatty alcohols produced may have a different fattyalcohol profile than the fatty alcohols produced by the control cell.Improved properties of FAR variants include, but are not limited to,increased total fatty alcohol production; increased production of fattyalcohols at a specified culture pH (e.g., pH 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, or 8) or over a broader pH range (e.g., pH 3.5-7);increased production of fatty alcohols at a specified culturetemperature (e.g., 30° C., 37° C., or 40° C.), or over a broadertemperature range (e.g., 30° C.-42° C.); and changes in fatty alcoholprofile as compared to a wild-type FAR. For example, a cell expressingthe improved FAR may have a fatty alcohol profile that includes a higherpercentage of total long chain fatty alcohols (e.g., C16-C18) orincluding a higher percentage of shorter chain fatty alcohols (e.g.,C12-C14). It will be understood that reference to a FAR variant “withincreased fatty alcohol production” and/or producing differential fattyacid profiles refers to fatty alcohol production by a host cellexpressing the FAR variant (e.g., host cells into which a recombinantpolynucleotide encoding the FAR variant has been introduced). In someembodiments the host cell is a yeast or bacteria. In some embodiments,the host cell is E. coli, which has proved particularly useful forproduction of fatty alcohols using the FAR variants disclosed herein.

Fatty Alcohol Production

In some embodiments, the FAR variants (or improved FAR polypeptides) ofthe invention, when expressed in a recombinant host cell, produce (i.e.,yield) an increased amount of fatty alcohols as compared to a wild-typeFAR having the sequence of SEQ ID NO:2 and/or a wild-type FAR having thesequence SEQ ID NO:5. In some embodiments, a FAR variant of the presentinvention has at least about 70% (or at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99%) sequence identity to SEQ ID NO:2 or SEQ ID NO:5 andcomprises one or more amino acid substitutions as described herein(e.g., one or more amino acid substitutions or amino acid substitutionsets listed in Table 2, Table 4, or Table 5), wherein a cell in whichthe FAR variant is expressed produces at least 1.5 times more fattyalcohol than a corresponding cell of the same type which expresses thewild-type FAR from which the FAR variant is derived under the sameconditions. In some embodiments, the fatty alcohol production of a hostcell (e.g., E. coli) expressing a FAR variant and a corresponding hostcell of the same type expressing the wild-type FAR from which the FARvariant is derived is measured under the same culture medium conditions(e.g., using LB or M9YE medium), the same temperature conditions (e.g.,at 30° C. or at 37° C.), and the same time conditions (e.g., for 24hours). In some embodiments the host cell (e.g., E. coli) expressing theFAR variant produces at least 2-fold, at least 3-fold, at least 4-fold,at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, atleast 9-fold, or at least 10-fold more fatty alcohols as compared to thecorresponding host cell of the same type expressing the wild-type FARfrom which the FAR variant is derived.

In some embodiments, the improved FAR polypeptides of the invention arecapable of producing more fatty alcohol than a wild-type polypeptidecorresponding to SEQ ID NO:2 when the polynucleotide encoding theimproved FAR polypeptide as described herein is expressed in a hostcell.

Fatty alcohol production is measured in a host cell, such as an E. colihost cell or a yeast (e.g., Yarrowia or Saccharomyces) host cell. SeeExamples 2, 4, and 6 below. In some embodiments, the improved FARpolypeptide of the invention is capable of producing more fatty alcoholthan a wild-type polypeptide corresponding to SEQ ID NO:2 when thepolynucleotide encoding the improved FAR polypeptide is expressed in E.coli and cultured under the same conditions. In some embodiments, theimproved FAR polypeptide of the invention is capable of producing morefatty alcohol than a wild-type polypeptide corresponding to SEQ ID NO:2when the polynucleotide encoding the improved FAR polypeptide isexpressed in a yeast and cultured under the same conditions. In oneembodiment, the yeast is Yarrowia lipolytica. In one embodiment, theyeast is Saccharomyces cerevisiae.

In some embodiments, the improved FAR polypeptide of the invention iscapable of producing more fatty alcohol than a wild-type polypeptidecorresponding to SEQ ID NO: 2 when total fatty alcohol production isdetermined. “Total fatty alcohol,” as used herein, refers to theintracellular and secreted amount of fatty alcohol. In some embodiments,the improved FAR polypeptide of the invention is capable of producingmore fatty alcohol than a wild-type polypeptide corresponding to SEQ IDNO:2 when secreted fatty alcohol production is determined. “Secretedfatty alcohol,” as used herein, refers to the extracellular fattyalcohol.

Fatty alcohol content can be measured using art known methods, such asmethods described herein below. In some embodiments, the improved FARpolypeptide of the invention is capable of producing more fatty alcoholthan a wild-type polypeptide corresponding to SEQ ID NO:2 when fattyalcohol production is determined by gas chromatography. In someembodiments, all fatty alcohols that are produced (e.g., all secretedfatty alcohols or total fatty alcohol) can be measured. In certainembodiments, total fatty alcohol production or secreted fatty alcoholproduction is determined by measuring production of representative fattyalcohols C14:0 (1-tetradecanol), C16:1 (cis Δ⁹-1-hexadecenol), C16:0(1-hexadecanol), C18:1 (cis Δ¹¹-1-octadecenol), and C18:0(1-octadecanol) produced.

Fatty Alcohol Profile

In some embodiments, the FAR variants of the invention produce fattyalcohol profiles that differ from the fatty alcohol profiles produced bywild-type FAR. In some embodiments, a FAR variant of the presentinvention has at least about 70% (or at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%; at least about 96%, at least about 97%, at least about 98%, or atleast about 99%) sequence identity to SEQ ID NO:2 or SEQ ID NO:5 andcomprises one or more amino acid substitutions as described herein(e.g., one or more amino acid substitutions or amino acid substitutionsets listed in Table 2, Table 4, or Table 5), wherein a cell in whichthe FAR variant is expressed produces a fatty alcohol profile thatdiffers from the fatty alcohol profile that is produced by acorresponding cell of the same type expressing the wild-type FAR fromwhich the FAR variant derived. when cultured under the same conditions.For example, a cell (e.g., E. coli) expressing the FAR variant mayproduce a higher percentage of a particular fatty alcohol than the cellexpressing the wild-type (e.g., a higher percentage of C14:0 or C16:1);or produce a lower percentage of a particular fatty alcohol than thecell expressing the wild-type (e.g., a lower percentage of C18:1); orproduces a higher percentage of a range of fatty alcohols than the cellexpressing the wild-type (such as a higher percentage of C10-C16 fattyalcohols). Generally, the fatty alcohol profiles produced by a host cell(e.g., E. coli) expressing a FAR variant and by a corresponding hostcell of the same type expressing the wild-type FAR from which the FARvariant is derived are measured by culturing the cells under the sameconditions, e.g, the same culture medium conditions (e.g., using LB orM9YE medium), the same temperature conditions (e.g., at 30° C. or at 37°C.), and for the same culture period conditions (e.g., culture for 24hours).

A fatty alcohol profile refers to the chain length distribution in acomposition containing fatty alcohols, or the chain length distributionof fatty alcohols produced by a cell. In some embodiments, the fattyalcohol profile contains saturated fatty alcohols, unsaturated fattyalcohols, or a mixture of saturated and unsaturated fatty alcohols. Thedegree of unsaturation and position within a chain can also vary. Forexample, a fatty alcohol may have 1, 2, 3, or more double bonds.Additionally, two fatty alcohols with the same chain length may eachhave a single double bond but at different positions. In someembodiments, the relative proportions of C14:0 (1-tetradecanol), C16:1(cis Δ⁹-1-hexadecenol), C16:0 (1-hexadecanol), C18:1 (cisΔ¹¹-1-octadecenol), and C18:0 (1-octadecanol) are measured to determinefatty alcohol profile. Fatty alcohol profiles can be measured using artknown methods, such as methods described herein below.

The fatty alcohol profiles produced by FAR variants of the presentinvention are beneficial for various purposes, including but not limitedto fuel compositions, detergent compositions, and cosmetic compositions.As is known in the art and described hereinbelow, fatty alcohols orfatty alcohol derivatives of specified chain length(s) are particularlyuseful in particular applications. Non-limiting examples include, forexample, fatty alcohols or fatty alcohol derivatives having a chainlength of C12-C14, which may be used in household care products; fattyalcohols or fatty alcohol derivatives having a chain length of C12-C18,which may be used in fuel compositions; and fatty alcohols or fattyalcohol derivatives having a chain length of C16-C18, which may be usedin personal care products.

As described in Example 3 and Table 3, below, fatty alcohol profile isaffected by the FAR variant selected, the background strain, and cultureconditions (e.g., temperature). For example, the fatty alcohol profileof FAR variant number 436 includes 19% C14:0 when expressed in E. coliBW25113-ΔtorR at 30° C., and 38% C14:0 when expressed in E. coliW3110-ΔfhuA at 37° C. Accordingly, fatty alcohol profiles that areproduced by the methods of the present invention can be influenced byadjusting conditions such as the FAR variant used, temperature, andbackground strain.

In some embodiments, the fatty alcohol compositions produced by themethods described herein have a fatty alcohol profile comprising atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, or atleast about 95% C10-C18 fatty alcohols. In some embodiments, the fattyalcohol compositions produced by the methods described herein have afatty alcohol profile comprising at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, or at least about 95% C10-C14 fattyalcohols. In some embodiments, the fatty alcohol compositions producedby the methods described herein have a fatty alcohol profile comprisingat least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,or at least about 95% C12-C16 fatty alcohols. In some embodiments, thefatty alcohol compositions produced by the methods described herein havea fatty alcohol profile comprising at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, or at least about 95% C14-C16 fattyalcohols. In some embodiments, the fatty alcohol compositions producedby the methods described herein have a fatty alcohol profile comprisingat least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,or at least about 95% C14-C18 fatty alcohols. In some embodiments, thefatty alcohol compositions produced by the methods described herein havea fatty alcohol profile comprising at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, or at least about 95% C16-C18 fattyalcohols. In some embodiments, the fatty alcohol compositions producedby the methods described herein have a fatty alcohol profile comprisingless than about 20%, less than about 15%, less than about 10%, or lessthan about 5% 018 fatty alcohols.

In some embodiments, the fatty alcohol compositions produced by themethods described herein have a fatty alcohol profile comprising amixture of 1-tetradecanol (C14:0), 1-hexadecanol (C16:0), 1-octadecanol(C18:0), cis Δ⁹-1-hexadecenol (C16:1), and/or cis Δ¹¹-1-octadecenol(C18:1) fatty alcohols. In some embodiments, the fatty alcohol profilecomprises at least about 10%, at least about 15%, at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60% or more of C14:0 fatty alcohol. In some embodiments, thefatty alcohol profile further comprises at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, or more of C16:1 fatty alcohol. In someembodiments, the fatty alcohol profile further comprises at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50% or more of C16:0 fatty alcohol. In someembodiments, the fatty alcohol profile further comprises up to about 5%,up to about 10%, up to about 15%, up to about 20%, or up to about 25% ofC18:1 fatty alcohol.

In some embodiments, the fatty alcohol composition further comprises oneor more of another C8-C20 fatty alcohol, for example, 1-octanol (C8:0),1-decanol (C10:0), 1-dodecanol (C12:0), 1-heptadecanol (C17:0),1-octadecanol (C18:0), or 1-icosanol (C20:0).

In some embodiments, the fatty alcohol composition has a fatty alcoholprofile comprising a mixture of 1-tetradecanol (C14:0), 1-hexadecanol(C16:0), 1-octadecanol (C18:0), cis Δ⁹-1-hexadecenol (C16:1), and/or cisΔ¹¹-1-octadecenol (C18:1) fatty alcohols in the following proportions(where the total percentage of fatty alcohols is 100%): about 10%-30%C14:0, about 30%-45% C16:1, about 30%-20% C16:0, and about 30%-5% C18:1.In some embodiments, the fatty alcohol composition has a fatty alcoholprofile comprising a mixture of 1-tetradecanol (C14:0), 1-hexadecanol(C16:0), 1-octadecanol (C18:0), cis Δ⁹-1-hexadecenol (C16:1), and/or cisΔ¹¹-1-octadecenol (C18:1) fatty alcohols in the following proportions:about 15%-35% C14:0, about 35%-45% C16:1, about 30%-15% C16:0, and about20%-5% 018:1. In some embodiments, the fatty alcohol composition has afatty alcohol profile comprising a mixture of 1-tetradecanol (C14:0),1-hexadecanol (C16:0), 1-octadecanol (C18:0), cis Δ⁹-1-hexadecenol(C16:1), and/or cis Δ¹¹-1-octadecenol (C18:1) fatty alcohols in thefollowing proportions: about 20%-30% C14:0, about 35%-25% C16:1, about30%-40% C16:0, and about 15%-5% C18:1. In some embodiments, the fattyalcohol composition has a fatty alcohol profile comprising a mixture of1-tetradecanol (C14:0), 1-hexadecanol (C16:0), 1-octadecanol (C18:0),cis Δ⁹-1-hexadecenol (C16:1), and/or cis Δ¹¹-1-octadecenol (018:1) fattyalcohols in the following proportions: about 20%-40% C14:0, about30%-40% C16:1, about 30%-20% C16:0, and about 20%-0% C18:1. In someembodiments, the fatty alcohol composition has a fatty alcohol profilecomprising a mixture of 1-tetradecanol (C14:0), 1-hexadecanol (C16:0),1-octadecanol (C18:0), cis Δ⁹-1-hexadecenol (C16:1), and/or cisΔ¹¹-1-octadecenol (C18:1) fatty alcohols in the following proportions:about 15%-30% C14:0, about 40%-45% C16:1, about 20%-25% C16:0, and about25%-0% C18:1. In some embodiments, the fatty alcohol composition has afatty alcohol profile comprising a mixture of 1-tetradecanol (C14:0),1-hexadecanol (C16:0), 1-octadecanol (C18:0), cis Δ⁹-1-hexadecenol(C16:1), and/or cis Δ¹¹-1-octadecenol (C18:1) fatty alcohols in thefollowing proportions: about 25%-30% C14:0, about 40%-45% C16:1, about25%-20% C16:0, and about 10%-5% C18:1. In some embodiments, the fattyalcohol composition has a fatty alcohol profile comprising a mixture of1-tetradecanol (C14:0), 1-hexadecanol (C16:0), 1-octadecanol (C18:0),cis Δ⁹-1-hexadecenol (C16:1), and/or cis Δ¹¹-1-octadecenol (C18:1) fattyalcohols in the following proportions: about 20%-30% C14:0, about35%-40% C16:1, about 35%-25% C16:0, and about 10%-5% C18:1. In someembodiments, the fatty alcohol composition has a profile that furthercomprises less than 5%, less than 4%, less than 3%, less than 2%, orless than 1% of C18:0.

In some embodiments, the fatty alcohol compositions produced by themethods described herein have a fatty alcohol profile comprising anincreased amount of C14:0 (1-tetradecanol), an increased amount of C16:1(cis Δ⁹-1-hexadecenol), a decreased amount of C16:0 (1-hexadecanol),and/or a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol) relative to awild-type FAR from which the FAR variant is derived (e.g., SEQ ID NO:2).In some embodiments, the fatty alcohol composition has a fatty alcoholprofile comprising at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 70%, at least80%, at least 90%, at least 100%, at least 150%, at least 200%, at least250%, at least 300%, at least 400%, at least 500% or more C14:0; atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50% ormore C16:1; at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50% or less C16:0; and at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 70%, atleast 80%, at least 90% or less C18:1 relative to the wild-type FAR fromwhich the FAR variant is derived.

In some embodiments, the fatty alcohol compositions produced by themethods described herein have a fatty alcohol profile comprising anincreased amount of C14:0 (1-tetradecanol), an increased amount of C16:1(cis Δ⁹-1-hexadecenol), an increased amount of C16:0 (1-hexadecanol),and/or a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol) relative to awild-type FAR from which the FAR variant is derived (e.g., SEQ ID NO:2).In some embodiments, the fatty alcohol composition has a fatty alcoholprofile comprising at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 70%, at least80%, at least 90%, at least 100%, at least 150%, at least 200%, at least250%, at least 300%, at least 400%, at least 500% or more C14:0; atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50% ormore C16:1; at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50% or more C16:0; and at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 70%, atleast 80%, at least 90% or less C18:1 relative to the wild-type FAR fromwhich the FAR variant is derived.

In some embodiments, the fatty alcohol compositions produced by themethods described herein have a fatty alcohol profile comprising anincreased amount of C14:0 (1-tetradecanol), a decreased amount of C16:1(cis Δ⁹-1-hexadecenol), an increased amount of C16:0 (1-hexadecanol),and/or a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol) relative to awild-type FAR from which the FAR variant is derived (e.g., SEQ ID NO:2).In some embodiments, the fatty alcohol composition has a fatty alcoholprofile comprising at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 70%, at least80%, at least 90%, at least 100%, at least 150%, at least 200%, at least250%, at least 300%, at least 400%, at least 500% or more C14:0; atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50% orless C16:1; at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50% or more C16:0; and at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 70%, atleast 80%, at least 90% or less C18:1 relative to the wild-type FAR fromwhich the FAR variant is derived.

Fatty Alcohol Measurements

FAR fatty alcohol production and fatty alcohol profiles (i.e., chainlength distribution) can be measured can be determined by methodsdescribed in the Examples section (e.g., Examples 2-4) and/or using anyother methods known in the art. Fatty alcohol production by an organismor culture expressing a FAR variant can be described as an absolutequantity (e.g., moles/liter of culture) or as a fold-improvement overproduction by an organism or culture expressing a reference FAR sequence(e.g., a wild-type FAR or a different FAR variant).

Fatty alcohol production and/or fatty alcohol profiles by amicroorganism expressing a FAR polypeptide can be measured, for example,using gas chromatography. In general, cells expressing a FAR variant arecultured, total or secreted fatty alcohols are isolated, and fattyalcohol amount and/or content is measured.

Any number of assays can be used to determine whether a host cellexpressing a FAR variant as described herein produces an increasedamount of fatty alcohols (e.g., at least 1.5 times more fatty alcohols)compared to a corresponding cell of the same type expressing a wild-typeFAR, and/or whether a host cell expressing a FAR variant as describedherein produces a different fatty alcohol profile compared to acorresponding cell of the same type expressing a wild-type FAR,including exemplary assays described herein. In one exemplary assay,fatty alcohols produced by productive E. coli strains are collected byextraction of 0.5 mL E. coli whole culture (culture medium plus cells)expressing a FAR variant using 1 mL of isopropanol:methyl t-butyl ether(MTBE) (4:6 ratio). The extraction mixture is allowed to shake for 2hours at room temperature. The extraction mixture is then centrifuged,the upper organic phase transferred into a vial and analyzed by the gaschromatography (GC) equipped with flame ionization detector (FID) andDB-5MS column (length 30 m, I.D. 0.32 mm, film 0.25 um), starting at150° C., and increasing the temperature at a rate of 25° C./min to 246°C., then holding for 1.81 min.

Fatty alcohol production by a host cell expressing a FAR variant canalso be compared to a comparable cell (“control cell”) expressing areference sequence, such as a wild-type FAR or a different FAR variant.Typically the FARs of the host and control cells are under control ofthe same promoter and the cells are maintained under the sameconditions. For illustration, fatty alcohol production can be measuredin E. coli (e.g., strain E. coli BW25113), using FARs under the controlof the same promoter (e.g., the lac promoter), where the cells arecultured at 37° C. and fatty alcohol produced after 24 hours of cultureare measured.

Fatty alcohol profiles (i.e., chain length distribution) can bedetermined, for example, using gas chromatography and/or massspectroscopy. In an exemplary assay, fatty alcohols are produced asdescribed above and the identification of individual fatty alcohols isperformed by comparison to commercial standards (Sigma Chemical Company,6050 Spruce St. Louis, Mo. 63103). The identity of the peaks can also beconfirmed by running the samples through a gas chromatography (GC)equipped with mass spectrometer (MS) as needed.

IV. Far Variants

In one aspect, the sequences of the improved FAR polypeptides describedherein include one or more mutations (e.g. substitutions) as compared toSEQ ID NO:2), such that the resulting FAR variant polypeptide hasimproved characteristics and/or properties as compared to the wild-typeFAR, such as, for example, increased fatty alcohol production. In arelated aspect, the invention provides a recombinant FAR variantcomprising one or more mutations (e.g. substitutions) as compared to awild-type FAR polypeptide from M. aquaeolei (SEQ ID NO:5), Substitutionsthat yield increased fatty alcohol production under these conditions aredescribed herein. These substitutions can be used singly or in anycombinations. In some embodiments, a FAR variant comprises a singlesubstitution set, e.g., a substitution set of any of Tables 2, 4, or 5.In some embodiments, a FAR variant comprises two or more substitutionsets. As is apparent from Table 2, combinations of substitutions can beselected so as to provide fatty alcohol production under the conditionsspecified that is anywhere from about 1.5-fold greater than that of thewild-type FAR of SEQ ID NO:2 to more than four-fold greater. In someembodiments, combinations of substitutions can be selected so as toprovide fatty alcohol production under the conditions specified that isat least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about10-fold greater than that of the wild-type FAR (e.g., SEQ ID NO:2 or SEQID NO:5).

Improved or increased fatty alcohol production of a FAR variant relativeto a reference polypeptide can be detected by comparing fatty alcoholproduction by host cells expressing the FAR variant to fatty alcoholproduction by host cells (of the same type) expressing the referenceprotein (which may be a wild-type or variant FAR) or a host cell notexpressing exogenous FAR. It will be understood by those of skill thatit is desirable that the only parameter varied between the cells is theFAR being expressed (e.g., a wild-type FAR or a FAR variant). Thus, forexample, the FAR variant and reference polypeptide will be encoded bypolynucleotides with the same sequence except at codons corresponding tosubstitutions (typically a sequence that is codon optimized for the celltype) and will be controlled by the same promoter, and cells expressingthe polypeptides will be cultured under the same conditions. Improved orincreased fatty alcohol production of a cell expressing a FAR variantrelative to a cell not expressing an exogenous FAR may also be measured.

For bacterial host cells, such as E. coli, exemplary assay conditionsare described in Examples 2, 3, and 4. In one approach, E. coli aretransformed with an expression cassette comprising a sequence encodingthe FAR variant or the reference protein (e.g., wild-type FAR, such asFAR Maa or FAR Maq) and an operably linked promoter. The cells may bestably transformed. The promoter may be constitutive or inducible. Thelac promoter may be used. The cells are grown in medium (e.g., M9YEmedium containing 0.5-5% glucose; Dunny, G. M., and Clewell, D. B.,1975. J. Bacteriol. 124:784-790) and any appropriate selection agents(see Examples). In one approach the cells are cultured for a period oftime (e.g., 18 hours or 24 hours) and fatty alcohol production isassayed. Typically, total fatty alcohol is assayed, but the amount offatty alcohol secreted into the medium may be assayed if desired. Insome embodiments, the promoter is inducible. For example. cells may begrown (e.g., to an OD₆₀₀ of 0.6-0.8), at which point expression of theheterologous FAR gene is induced (e.g., by addition of IPTG to a 1 mMfinal concentration when the lac promoter is used). Incubation iscontinued for 24 hours at 30° (alternatively 37° C. or 40° C.) and fattyalcohol production is assayed.

It will be apparent that the same assays may be used to assess FARactivity of functionally active fragments of FAR variants.

It has been discovered that certain substitutions, both singly and invarious combinations, yield improvements in fatty alcohol productioncompared to the wild-type FAR (see Tables 2 and 4). These substitutionsmay include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more than 20substitutions. In some embodiments, the improved FAR polypeptides of theinvention differ from SEQ ID NO:2 in up to 20 residues. In someembodiments, the improved FAR polypeptides of the invention differ fromSEQ ID NO:5 in up to 20 residues. In some embodiments, the improved FARpolypeptides differ from SEQ ID NO:2, or differ from SEQ ID NO:5, in upto 15 residues, sometimes in up to 12 residues, and sometimes in up to10 residues.

As shown in Table 2, it can be seen that substitutions introduced atnumerous different amino acid (also referred to herein as “residue”)positions in the wild-type FAR of SEQ ID NO:2 yield improved FARpolypeptides capable of producing increased yields of total fatty acidas compared to this wild-type FAR. Similarly, as shown in Table 4,substitutions in SEQ ID NO:5 increase fatty alcohol yield. Single aminoacid changes at specified positions give rise to 2-fold or greaterincreases in fatty alcohol production.

Accordingly, in some embodiments, the improved FAR polypeptide of theinvention contains at least one amino acid substitution relative to SEQID NO:2 and is capable of producing at least about 1.5-fold more fattyalcohol than a wild-type FAR corresponding to SEQ ID NO:2 when assayedunder the same conditions. In some embodiments, the improved FARpolypeptide is capable of producing at least about 1.5 more fattyalcohol than a wild-type FAR consisting of SEQ ID NO:2 when assayedunder the same conditions. In some embodiments, the improved FARpolypeptide of the invention contains at least one amino acidsubstitution relative to SEQ ID NO:5 and is capable of producing atleast about 1.5-fold more fatty alcohol than a wild-type FARcorresponding to SEQ ID NO:5 when assayed under the same conditions.

While single substitutions are beneficial, it can be seen from theexamples (e.g., Tables 2, 4, and 5) that combinations of substitutionscan yield further benefit. Thus, combinations of substitutions can beselected to yield improved FAR enzymes capable of producing specifiedlevels of total fatty alcohol when cultured under the conditionsdescribed herein (e.g., in Example 4). For example, improved FARpolypeptides can be readily designed that yield from 1.5, 1.8, 2.0, 2.2,2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.5 or even higher-foldincreases in total fatty alcohol compared to the wild-type FAR of SEQ IDNO:2. For example, in some embodiments, FAR variants can be readilydesigned that produce about 1.5, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2,3.4, 3.6, 3.8, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0,9.5, 10.0 or even higher-fold increases in total fatty alcohol comparedto the wild-type FAR of SEQ ID NO:2 or SEQ ID NO:5.

Accordingly, in some embodiments, a FAR variant of the present inventioncontains at least two, at least three, at least four, at least five, ormore amino acid substitutions relative to wild-type FAR (e.g., SEQ IDNO:2 or SEQ ID NO:5) and is capable of producing at least about1.5-fold, at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 6-fold, at least about7-fold, at least about 8-fold, at least about 9-fold, or at least about10-fold more fatty alcohol than the wild-type FAR (e.g., SEQ ID NO:2 orSEQ ID NO:5). In some embodiments, the improved FAR polypeptide containsat least two amino acid substitutions relative to SEQ ID NO:2 (or SEQ IDNO:5) and is capable of producing at least about 1.5-fold, at leastabout 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, atleast about 3.5-fold, at least about 4.0-fold more fatty alcohol than awild-type FAR corresponding to SEQ ID NO:2 (or SEQ ID NO:5) when assayedunder the same conditions. In some embodiments, the improved FARpolypeptide contains at least three amino acid substitutions relative toSEQ ID NO:2 (or SEQ ID NO:5) and is capable of producing at least about1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at leastabout 3.0-fold, at least about 3.5-fold, at least about 4.0-fold morefatty alcohol than a wild-type FAR corresponding to SEQ ID NO:2 (or SEQID NO:5) when assayed under the same conditions. In some embodiments,the improved FAR polypeptide contains at least four amino acidsubstitutions relative to SEQ ID NO:2 (or SEQ ID NO:5) and is capable ofproducing at least about 1.5-fold, at least about 2.0-fold, at leastabout 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, atleast about 4.0-fold more fatty alcohol than a wild-type FARcorresponding to SEQ ID NO:2 (or SEQ ID NO:5) when assayed under thesame conditions. In some embodiments, the improved FAR polypeptidecontains at least five amino acid substitutions relative to SEQ ID NO:2(or SEQ ID NO:5) and is capable of producing at least about 1.5-fold, atleast about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold,at least about 3.5-fold, at least about 4.0-fold more fatty alcohol thana wild-type FAR corresponding to SEQ ID NO:2 (or SEQ ID NO:5) whenassayed under the same conditions.

In some embodiments, a FAR variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 and comprises an amino acid substitution relative to SEQ ID NO:2at one or more positions selected from A2, T3, Q4, Q5, Q6, Q7, N8, G9,A10, A12, G14, E17, Q18, K22, V24, L33, I42, G50, L54, R60, H61, P62,A63, R65, L69, E71, A73, S74, S76, V77, H83, E87, T91, L93, H98, T101,G102, V104, S107, G110, L111, T112, P113, R115, R117, A120, G121, Q122,A125, N128, S132, N134, E137, E138, D140, A142, K144, L148, E151, V153,N160, A162, N174, N177, Q180, V185, 1186, P188, T197, D198, E202, E204,E205, V207, L209, D212, K213, V217, R220, K224, L226, E227, K229, R236,E237, S244, D245, T246, L257, K260, A261, S263, G264, S266, 1269, S283,I287, E288, V290, A295, A299, E303, V305, S306, V318, I328, L330, S331,L332, A333, S339, G340, Q341, R342, G350, G351, K359, L364, M365, A366,T370, A374, D376, Q377, Y380, R381, T384, A389, D396, V397, V398, V399,G400, G401, R403, V404, P405, L406, A409, G410, A412, M413, A416, Q418,E421, N427K, D429, T430, R432, S433, T436, 1437, F440, A443, P444, Y446,S452, S458, R459, L463, D464, V466, A472, 0474, L479, 1484, G487, N490,E496, K498, L499, Y500, S501, L502, A504, A505, D506, T507, 8508, K509,K510, A511, and A512, wherein the position is numbered with reference toSEQ ID NO:2. In some embodiments, the variant comprises one or moreamino acid substitutions selected from A2D/F/G/H/I/P/N/Q/T/V/W, T3R,Q4R, Q5S, Q6P, Q7N, N8K/S, G9D/F, A10T, A12T/V, G14N/R/V/W, E17D, Q18I,K22E, V24I, L33V, I42L, G50S/V, L54P, R60H, H61R, P62S, A63R/Y,R65G/Q/Y, L69E/Q, E71K, A73K/V, S74K/P, S76K/N/R, V77A/I, H83R, E87G/V,T91I/R, L93V, H98P/R, T101L, G102C, V104I/M, S107C/L/W, G110D, L111S,T112A, P113D/L, R115A/H, R117D, A120V, G121H/S, Q122R, A125V, N128H,S132G, N134K/R/S, E137L, E138L/Q, D140C, A142V, K144Q, L148E, E151L,V153I, N160S, A162T, N174C, N177Q/R/T, Q180H/R, V185A/I, I186A/G/Y,P188A/I/M/S, T197P, D198Q, E202G, E204G, E205G/P, V207I/L, L209K/N,D212R, K213R, V217L, R220C, K224R, L226A/M, E227A/G/H/R/T, K229R, R236K,E237L, S244A/F/G/H/P, D245N, T246A, L257K, K260R/T, A261D, S263P, G264S,S266A, I269T, S283E/F/K/M/T/V, I287L, E288Q, V290I, A295T/V, A299T,E303G, V3051, S306F/H/N/W, V318I, I328T, L330V, S331V, L332S, A333T,S339G/V, G340P/S/V, Q341K, R342L, G350S, G351C, K359L, L364F/I, M365N,A366T/V, T370A/I, A374K/Y, D376P, Q377C/K/Y, Y380K/N/R, R381C, T384R,A389I/L/M/V, D396G, V397I/L, V398Y, V399T, G400A/L, G401A/C/I/L/S/V,R403C/S, V404A, P405A/C/F/G/L/S/V/W, L406Y, A409V/W/Y,G410A/C/H/N/Q/R/S, A412C/F/M/V, M413L/R, A416L/V, Q418I/R/V/Y,E421I/L/N/P/R/S/V/Y, N427K, D429E/K/N/Q/R, T430H/I/R, R432C/Q,S433F/H/K/L/N/W, T436D/K/Q, I437V, F440L, A443T, P444S, Y446H,S452A/G/N, S458G/L/M/Q, R459H, L463E/T, D464G, V466E/Q/R, A472V, Q474R,L479Q, I484V, G487R/S/T/Y, N490S, E496A, K498A, L499A/H/I/N/P/R/S,Y500C/G/H/L/N/P/Q/R/S/W, S501G/R, L502A/P/Q/R/S, A504G/R, A505K,D506G/S, T507A/G/P/R/S, R508D/G/H, K509D/E/G/H/N/R/S/Y, K510A/D/G/P/S/Y,A511G/I/K/P/Q/R/S/T, and A512K/S/T.

In some embodiments, a FAR variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 and comprises a substitution relative to SEQ ID NO:2 at one ormore positions selected from position 2, position 134, position 138,position 140, position 421, position 458, position 510, and position511, wherein the position is numbered with reference to SEQ ID NO:2. Theresidues at positions 2, 134, 138, 140, 421, 458, 510, and 511 have beenidentified by the inventors as being important for fatty alcoholproduction, as shown in the Examples below. As shown below and in FIG.2, positions 2, 134, 138, 140, 421, 458, 510, and 511 of SEQ ID NO:2correspond to positions 2, 135, 139, 141, 422, 459, 511, and 512,respectively, of SEQ ID NO:5. In some embodiments, the amino acidresidue at position 2 relative to SEQ ID NO:2 is alanine (A2); the aminoacid residue at position 134 relative to SEQ ID NO:2 is asparagine(N134); the amino acid residue at position 138 relative to SEQ ID NO:2is glutamic acid (E138); the amino acid residue at position 140 relativeto SEQ ID NO:2 is aspartic acid (D140); the amino acid residue atposition 421 relative to SEQ ID NO:2 is glutamic acid (E421); the aminoacid residue at position 458 relative to SEQ ID NO:2 is serine (S458);the amino acid residue at position 510 relative to SEQ ID NO:2 is lysine(K510); and the amino acid residue at position 511 relative to SEQ IDNO:2 is alanine (A511). In some embodiments, a FAR variant comprises anamino acid substitution at residue A2 that is selected from asparticacid, phenylalanine, glycine, histidine, isoleucine, asparagine,proline, glutamine, threonine, valine, or tryptophan(A2D/F/G/H/I/N/P/Q/T/V/W). In some embodiments, a FAR variant comprisesan amino acid substitution at residue N134 that is selected from lysine,arginine, or serine (N134K/R/S). In some embodiments, a FAR variantcomprises an amino acid substitution at residue E138 that is selectedfrom leucine or glutamine (E138L/Q). In some embodiments, a FAR variantcomprises an amino acid substitution at residue D140 that is cysteine(D140C). In some embodiments, a FAR variant comprises an amino acidsubstitution at residue E421 that is selected from isoleucine, leucine,asparagine, proline, arginine, serine, valine, or tyrosine(E421I/L/N/P/R/S/V/Y). In some embodiments, a FAR variant comprises anamino acid substitution at residue S458 that is selected from glycine,leucine, methionine, or glutamine (S458G/L/M/Q). In some embodiments, aFAR variant comprises an amino acid substitution at residue K510 that isselected from alanine, aspartic acid, glycine, proline, serine, ortyrosine (K510A/D/G/P/S/Y). In some embodiments, a FAR variant comprisesan amino acid substitution at residue A511 that is selected fromglycine, isoleucine, lysine, proline, glutamine, arginine, serine, orthreonine (A511G/I/K/P/Q/R/S/T).

It is notable that positions 511 of SEQ ID NO:2 and 512 of SEQ ID NO:5are very close to the carboxy-terminus of the FAR protein. Indeed,substitutions at one or more of residues 501-511 (numbered accoding toSEQ ID NO:2) are found in many FAR variants that support increased fattyalcohol production, and substitutions at 501, 502, 504, 507, 509, 510,and 511 were found in both M. algicola FAR and M. aquaeolei FAR (seeTable 4A). These residues are in the “Sterile” pfam domain (see, e.g.,the website pfam.sanger.ac.uk/). Further, without intending to be boundby a particular mechanism, the C-termini of M algicola FAR and M.aquaeolei FAR (RKKAA and RKKAA) are characterized by the motif +++HHwhere “+” is a positively charged amino acid (e.g., K, R) and “H” is ahydrophobic amino acid (e.g., A, I, L, F, V, P and G). Thus, in oneaspect a FAR variant of the invention has at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 93%,at least about 95%, at least about 97%, and at least about 98% identityto a wild-type FAR (e.g., SEQ ID NO: 2 and/or SEQ ID NO: 5) andcomprises a C-terminal amino acid sequence that is not the +++HH motif.

In some embodiments, the FAR variant further comprises a substitutionrelative to SEQ ID NO:2 at one or more of position 188, position 405,and position 418, wherein the positions are numbered with reference toSEQ ID NO:2. The residues at positions 188, 405, and 418 have beenidentified by the inventors as being important for fatty alcoholproduction, as shown in the Examples below. As shown below and in FIG.2, positions 188, 405, and 418 of SEQ ID NO:2 correspond to positions189, 406, and 419, respectively, of SEQ ID NO:5. In some embodiments,the amino acid residue at position 188 relative to SEQ ID NO:2 isproline (P188); the amino acid residue at position 405 relative to SEQID NO:2 is proline (P405); and the amino acid residue at position 418relative to SEQ ID NO:2 is glutamine (Q418). In some embodiments, a FARvariant comprises an amino acid substitution at residue P188 that isselected from alanine, isoleucine, methionine, or serine (P188A/I/M/S).In some embodiments, a FAR variant comprises an amino acid substitutionat residue P405 that is selected from alanine, cysteine, phenylalanine,glycine, leucine, serine, valine, or tryptophan (P405A/C/F/G/L/S/V/W).In some embodiments, the FAR variant comprises an amino acidsubstitution at residue Q418 that is selected from isoleucine, arginine,valine, and tyrosine (Q418I/R/V/Y).

In some embodiments, improved FAR polypeptides comprise at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% identity to the wild-type FAR ofSEQ ID NO:2, that include substitutions at one or more of positions 2,134, 138, 188, 405, 418 and 511 when aligned to SEQ ID NO:2. As shownbelow and in FIG. 2, positions 2, 134, 138, 188, 405, and 418 correspondto positions 2, 135, 139, 189, 406, and 419, respectively, of SEQ IDNO:5. In some embodiments, the substitution at position 2 is H, T, D, F,V, G, Q, P or I (particularly G, F, Q and D); the substitution atposition 134 is R, K or S (particularly S); the substitution at position138 is Q or L (particularly Q); the substitution at position 188 is S;the substitution at position 405 is any amino acid; preferably V, S, F,G, C, L, S, A, W (particularly an aliphatic amino acid); thesubstitution at position 418 is V, R, I or Y; and the substitution atposition 511 is any amino acid and particularly T, P, G, S, K, Q or R,and more particularly T and Q. In some embodiments the improved FAR willcomprises a sequence comprising at least 85%, at least 90%, at least95%, and at least 97% sequence identity to SEQ ID NO: 2 and asubstitution at position 134 and 511 when aligned with SEQ ID NO: 2. Insome embodiments, the improved FAR enzyme will comprise additionalsubstitutions at one or more positions 303, 401, 416, 499, 505, 508,509, 510 when aligned to SEQ ID NO:2. In some particular embodimentssuch optional additional substitutions include one or more of thefollowing substitutions (relative to SEQ ID NO:2): 303G, 401 (aliphaticamino acid or S); 418 (aliphatic amino acid or R; preferably I or V),416 (V or L; preferably L), 499 (R, P, S, H, N, I, or A), 505K, 508G and509 (G, H, E, R, D, S or N) and 510 (S, D or Y).

In some embodiments, the improved FAR polypeptides comprise at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99% identity to the wild-type FARof SEQ ID NO:2, that include substitutions at one or more of positionsA2, N134, E138, P188, P405, Q418 and A511 when aligned to SEQ ID NO:2.In some embodiments, the substitution at position 2 is H, T, D, F, V, G,Q, P or I (particularly G, F, Q and D); the substitution at position 134is R, K or S (particularly S); the substitution at position 138 is Q orL (particularly Q); the substitution at position 188 is S; thesubstitution at position 405 is any amino acid; preferably V, S, F, G,C, L, S, A, W (particularly an aliphatic amino acid); the substitutionat position Q418 is V, R, I or Y; and the substitution at position A511is any amino acid and particularly T, P, G, S, K, Q or R, and moreparticularly T and Q. In some embodiments the improved FAR willcomprises a sequence comprising at least 85%, at least 90%, at least95%, and at least 97% sequence identity to SEQ ID NO:2 and asubstitution at position N134 and A511 when aligned with SEQ ID NO: 2.In some embodiments, the improved FAR enzyme will comprise additionalsubstitutions at one or more of positions E303, G401, A416, L499, L502,A505, R508, K509, and K510 when aligned to SEQ ID NO:2. In someparticular embodiments such optional additional substitutions includeone or more of the following substitutions (relative to SEQ ID NO:2):E303G, G401 (aliphatic amino acid or S); Q418 (aliphatic amino acid orR; preferably I or V), A416 (V or L; preferably L), L499 (R, P, S, H, N,I, or A), L502 (S, Q, A, R, or P), A505K, R508G and K509 (G, H, E, R, D,S or N) and 510 (S, D or Y). The additional substitutions may also beselected from any of the substitutions in Table 2.

In some embodiments, the improved FAR polypeptide comprises a sequencehaving at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% sequence identity tosequence SEQ ID NO:2 and a substitution at position 511. In particularembodiments, the improved FAR polypeptide comprises an amino acidsubstitution at position 511: T, P, G, S, K, Q, or R relative to SEQ IDNO:2.

In some embodiments, the improved FAR polypeptide comprises thefollowing amino acid substitutions relative to SEQ ID NO:2: positionN134: R, K or S; position E138: Q or L; position P188: S: and positionA511: T, P, G, S, K, Q and R. In other particular embodiments, theimproved FAR enzyme comprises the following amino acid substitutionsrelative to SEQ ID NO:2; position 134: S; position 138: L; position 188:S; and position 511: T.

In some embodiments, the improved FAR polypeptide comprises thefollowing amino acid substitution relative to SEQ ID NO:2: a) position134 and position 188 and particularly position 134 is S and position 188is S; b) position 134, position 138 and position 188, and particularlyposition 134 is S; position 138 is Q; and position 188 is S; c) position188 and position 511, and particularly position 188 is S and position511 is T; d) position 134, position 138, position 188, position 405 andposition 511 and particularly 134 is S; 138 is Q; 188 is S; 405 is V and511 is T. The improved FAR polypeptide may include further substitutionssuch as 1, 2, 3, 4, 5, 6, 7 or more substitutions relative to SEQ IDNO:2. The one or more substitutions may be selected from positions 2,303, 401, 416, 418, 499, 502, 505, 508, or 509 relative to SEQ ID NO:2,and more particularly 2 (D, V, F, T, N, H, W, P, I, Q, or G), 303 (G),401 (V, I, L, S, or A) 412 (V, F or C), 416 (L or V), 418 (R, V, I, orY), 499 (R, P, S, H, N, I, or A), 502 (S, Q, A, R, or P), 505 (K), 508(G, H or D), or 509 (D, H, S, E, G, N, R or Y) relative to SEQ ID NO:2.

In some embodiments, the improved FAR polypeptide comprises an aminoacid substitution at one or more positions corresponding to position 2,134, 140, 107, 237, 410, 421, 429, 433, 458, 499, 502, 504, 509, or 511of SEQ ID NO:2 or a sequence having at least 75%, at least 80%, at least85%, at least 90%, at least 93%, at least 95%, at least 97%, or at least98% identity with SEQ ID NO:2 and optionally at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, or at least 8further substitutions.

In some embodiments, a FAR variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:5 and comprises an amino acid substitution relative to SEQ ID NO:5at one or more positions selected from A2, Q4, Q5, H8, A9, A45, P63,R66, E72, A74, S77, E88, A108, G111, G113, D116, N135, D141, Q181, D199,E205, E238, A374, A375, P406, D411, R412, D422, D430, S434, 1438, N459,E497, Y501, S502, L503, T505, Q508, R509, K510, K511, A512, and A513,wherein the position is numbered with reference to SEQ ID NO:5. In someembodiments, the variant comprises one or more amino acid substitutionsselected from A2F/G/H/P/Q/T, Q4I, Q5F/N, H8K/N, A9L, A45V, P63Q/S, R66N,E72Q/S, A74L, S77G, E88Q, A108C/L/R, G111S, G113A, D116A/E, N135K,D141C/G, Q181D, D199G, E205G/R, E238C, A374V, A375Q/Y, P406S, D411R,R412H, D422A, D430K, S434F/K/W, 1438V, N459G/Q, E497F/Y, Y501 G/P/S/W,S502G, L503Q/R/S, T505K/R, Q508G/S, R509A/D, K510G, K511C/D/G,A512G/K/P/Q/S/T, and A513L/Y.

In some embodiments, a variant as described herein is encoded by apolynucleotide that hybridizes at high stringency to the complement ofSEQ ID NOs:1, 3, 4, 13 or 14 and comprises one or more amino acidsubstitutions as described herein.

In some embodiments, a FAR variant comprising any of the mutationsdescribed herein (e.g., any of the FAR variants in Tables 2, 4, and 5 aswell as any variants that comprise an amino acid substitution setprovided in Table 2, Table 4, or Table 5) comprises at least at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity to a wild-type FAR producedby M. algicola (e.g., SEQ ID NO:2). In some embodiments, a FAR variantcomprising any of the mutations described herein (e.g., any of the FARvariants in Tables 2, 4, and 5 as well as any variants that comprise anamino acid substitution set provided in Table 2, Table 4, or Table 5)comprises at least at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identityto a wild-type FAR produced by M. aquaeolei (e.g., SEQ ID NO:5).

Any of the improved FAR polypeptides as described herein may alsoinclude one or more additional substitutions, or one or more insertionsor deletions, in addition to the specified substitutions describedherein. It is expected that FAR polypeptides having at least about 75%(e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%) sequence identity with the wild-typeFAR of SEQ ID NO:2 or SEQ ID NO:5 and also having one or more of thespecified substitutions will exhibit improved characteristics and/orproperties as described herein.

In some embodiments, the improved FAR polypeptides may have less thanabout 99%, less than about 98%, less than about 97%, less than about96%, less than about 95%, less than about 93%, less than about 90%, orless than about 85% sequence identity to SEQ ID NO:2 or SEQ ID NO:5.

Residue positions which have been found to be important to total fattyalcohol yield, and which provide significant increases when used invarious different combinations, include residue positions correspondingto positions 134, 138, 188 and 511 of SEQ ID NO:2. The wild-typeresidues at these positions may be substituted by any one of the othernaturally encoded amino acids. In some embodiments, the substitutions atthese positions are, independently of one another, selected from thefollowing: position 134: R, K or S (preferably S); position 138: Q or L(preferably L); position. 188: S; and position 511: T, P, G, S, K, Q, orR (preferably T).

In some embodiments, the improved FAR polypeptides contain an amino acidsubstitution at position 134 relative to SEQ ID NO:2. In someembodiments, the improved FAR polypeptide contains an amino acid R, K,or S at position N134 relative to SEQ ID NO:2.

In some embodiments, the improved FAR polypeptides contain an amino acidsubstitution at position 138 relative to SEQ ID NO:2. In someembodiments, the improved FAR polypeptide contains an amino acid 0 or Lat position E138 relative to SEQ ID NO:2.

In some embodiments, the improved FAR polypeptides contain an amino acidsubstitution at position P188 relative to SEQ ID NO:2. In someembodiments, the improved FAR polypeptide contains an amino acid S atposition 188 relative to SEQ ID NO:2.

In some embodiments, the improved FAR polypeptides contain an amino acidsubstitution at position P405 relative to SEQ ID NO:2. In someembodiments, the improved FAR polypeptide contains an amino acid V, S,F, G, C, L, S, A, or W at position 405 relative to SEQ ID NO:2.

In some embodiments, the improved FAR polypeptides contain an amino acidsubstitution at position A511 relative to SEQ ID NO:2. In someembodiments, the improved FAR polypeptide contains an amino acid T, P,G, S, K, Q, or R at position 511 relative to SEQ ID NO:2. In preferredembodiments, the polypeptide contains the amino acid T at position 511relative to SEQ ID NO:2.

In some embodiments, the polypeptide contains substitutions for at leasttwo of positions N134, E138, P188 and A511 relative to SEQ ID NO:2. Inother embodiments, the polypeptide contains substitutions for at leastthree of positions N134, E138, P188 and A511 relative to SEQ ID NO:2. Inother embodiments, the polypeptide contains substitutions for all fourof positions 134, 138, 188 and 511 relative to SEQ ID NO:2. In theseembodiments, wherein the polypeptide comprises substitutions for atleast two, at least three, or all four, of positions 134, 138, 188 and511 relative to SEQ ID NO:2, the substitutions may independently beselected from the following: position 134: R, K or S (preferably S);position 138: Q or L (preferably L); position 188: S; and position 511:T, P, G, S, K, Q, or R (preferably T).

In preferred embodiments, the improved FAR polypeptide of the inventioncontains the following amino acid substitutions relative to SEQ ID NO:2:position 134: R, K or S; position 138: Q or L; position 188: S; andposition 511: T, P, G, S, K, Q, R.

In other preferred embodiments, the improved FAR polypeptide of theinvention contains the following amino acid substitutions relative toSEQ ID NO:2: position 134: S; position 138: L; position 188: S; andposition 511:T.

One or more additional substitutions may also be beneficial. Forexample, it has been found that including one or more additionalsubstitutions at residue positions corresponding to residue positions303, 401, 405, 416, 418, 505, 508 and 509 of SEQ ID NO:2 yieldbeneficial increases in fatty alcohol yield. When such additionalsubstitutions are included in an improved FAR enzyme, the wild-typeresidue may be mutated to any one of the other naturally encoded aminoacids. In some embodiments, the substitutions at these positions are,independently of one another, selected from the following: position303:G; position 401:aliphatic amino acid (e.g., V, L, A I) or S(preferably aliphatic amino acid); position 405:aliphatic amino acid, G,C or F (preferably aliphatic amino acid); position 416: aliphatic aminoacid (preferably V or L); position 418: aliphatic amino acid, R or Y(preferably Y); position 505: basic amino acid (preferably K); position508: G, H (preferably H); position 509: G, H, E, R, D, S, or N(preferably D).

In some embodiments, the improved FAR polypeptides have amino acidsequences corresponding to SEQ ID NO:2 that include the followingsubstitutions (relative to SEQ ID NO:2): N134 (S or R), E138Q, P188S andA511 (G, K, R or T), and optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore additional substitutions at any other residue position. Specificexemplary embodiments of this class of improved FAR polypeptides includevariant Nos: 211, 216, 238-264, and 266-423 of Table 2.

In some embodiments, a FAR variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to SEQID NO:2 and comprises a substitution relative to SEQ ID NO:2 at one ormore positions selected from K22, E87, N134, E138, P188, L209, G264,E303, G401, P405, A412, A416, Q418, S458, Y500, L502, 8508, K509, andA511, wherein the position is numbered with reference to SEQ ID NO:2.Substitutions at positions K22, E87, N134, E138, P188, L209, G264, E303,G401, P405, A412, A416, Q418, S458, Y500, L502, R508, K509, and A511, asnumbered with reference to a wild-type M. algicola FAR polypeptide(e.g., SEQ ID NO:2), were identified by the inventors as beingbeneficial for increasing fatty alcohol production or increasingproduction of a particular fatty alcohol profile.

Certain FAR variants of the present invention have an amino acidsequence that includes at least one amino acid substitution at one ormore amino acid residues selected from N134, E138, P188, S458, and A511,wherein the amino acid residues are numbered with reference to SEQ IDNO:2. Amino acid substitutions at one or more of these positions arepredicted to be beneficial substitutions for increasing fatty alcoholproduction or increasing production of a particular fatty alcoholprofile. In some embodiments, a FAR variant of the present invention hasan amino acid sequence that comprises at least about 70% (or at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%) sequence identity to SEQ IDNO:2 and comprises one or more amino acid substitutions selected fromN134R, E138Q, P188S, S458Q, and A511T, wherein the position is numberedwith reference to SEQ ID NO:2, which are predicted to be beneficialsubstitutions for increasing fatty alcohol production.

Certain FAR variants of the present invention have an amino acidsequence that includes at least one amino acid substitution at one ormore amino acid residues selected from E303, G401, P405, A412, A416,Q418, S458, L502, R508, and K509, wherein the amino acid residues arenumbered with reference to SEQ ID NO:2. Amino acid substitutions at oneor more of these positions are predicted to be beneficial substitutionsfor increasing fatty alcohol production or increasing production of aparticular fatty alcohol profile. In some embodiments, a FAR variant ofthe present invention has an amino acid sequence that comprises at leastabout 70% (or at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%)sequence identity to SEQ ID NO:2 and comprises one or more amino acidsubstitutions selected from E303G, G401A/L/S/V, P405A/C/F/L/V, A412V,A416L, Q4181/V, S458Q, L502S, R508G/H, and K509D/H, wherein the positionis numbered with reference to SEQ ID NO:2, which are predicted to bebeneficial substitutions for increasing fatty alcohol production.

Certain FAR variants of the present invention have an amino acidsequence that includes at least one amino acid substitution at one ormore amino acid residues selected from K22, E87, L209, G264, G401, A416,Y500, R508, and K509, wherein the amino acid residues are numbered withreference to SEQ ID NO:2. Amino acid substitutions at one or more ofthese positions are predicted to be beneficial substitutions forincreasing fatty alcohol production or increasing production of aparticular fatty alcohol profile. In some embodiments, a FAR variant ofthe present invention has an amino acid sequence that comprises at leastabout 70% (or at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%)sequence identity to SEQ ID NO:2 and comprises one or more amino acidsubstitutions selected from K22R, E87G, L209K/L/N, G264S, G401V, A416L,Y500D, R508D/G, and K509D/H/N/Y, wherein the position is numbered withreference to SEQ ID NO:2, which are predicted to be beneficialsubstitutions for increasing fatty alcohol production.

Functional Fragments of FAR Variants

Skilled artisans will appreciate that oftentimes, the full lengthsequence of an enzyme is not required for enzymatic activity. Therefore,“functional fragments” of the various improved FAR enzymes describedherein are also contemplated and included in the disclosure. ImprovedFAR enzymes and functional fragments thereof are sometimes referred toherein as “improved FAR polypeptides.” The enzymatic activity offunctional fragments may be measured as described in the Exampleshereinbelow.

In some embodiments the functional fragments comprise at least 85%, atleast 90%, at least 95%, and at least 98% of the corresponding improvedfull-length FAR enzyme. In many instances, functional fragments, likethe improved full-length FAR enzyme from which they are derived, willproduce 1.5-fold or higher total fatty alcohol than the wild-type FAR ofSEQ ID NO:2, when assayed under the same conditions. In someembodiments, the improved FAR polypeptide of the invention is from about350 to about 550 amino acids in length, e.g., from about 350 to about400 amino acids in length. In some embodiments, the improved FARpolypeptide of the invention is from 400 to 550 amino acids in length,such as from 400 to 500, from 450 to 500, from 500 to 550, from 500 to525, or from 505 to 515 amino acids in length.

In some embodiments, regions of one or both termini, such as, forexample, from about 1 to about 10; 1 to about 15; or 1 to about 20residues at one or both termini, may be removed without significantlydeleteriously affecting the activity of the enzyme. Such deletions canoften times be made internally without detrimental effect. In instancesof multifunctional, multi-domain enzymes, entire domains can be removedwithout deleteriously affecting a desired enzymatic activity.

Exemplary Substitutions in Marinobacter Algicola FAR Homologs

In another aspect, the present invention provides FAR proteins that arevariants of naturally occurring FAR enzymes of marine proteobacteriaspecies other than Marinobacter algicola (strain DG893) which comprise asubstitution or modification at at least one position corresponding to asubstitution of a M. algicola FAR variant described herein, and whichhave improved properties relative to the naturally occurring FAR enzyme.

In particular, analogous substitutions may be made in marinegammaproteobacteria with significant sequence similarity to Marinobacteralgicola (strain DG893). For example, analogous substitutions may bemade in other species of Marinobacter including but not limited to M.algicola, M. aquaeolei, M. arcticus, M. actinobacterium, and M.lipolyticus; species of Oceanobacter including but not limited toOceanobacter sp. Red65 (renamed Bermanella marisrubi), Oceanobacterstrain WH099, and O. kriegii; and species of Hahella including but notlimited to H. chejuensis and equivalent species thereof.

Improved FAR polypeptides may also be derived from FAR enzymesidentified from wild-type organisms using multiple sequence alignmentsusing Hidden Markov Models (“HMMs”), which identify proteins in compiledprotein family databases that share common domains withpreviously-identified suitable FAR enzymes. See, e.g., the websitepfam.sanger.ac.uk/. In certain embodiments, the HMMs are used toidentify NAD binding domains and/or sterile domains.

It is within the ability of one of ordinary skill in the art to identifyother examples of structurally homologous proteins. The presentinvention provides variants of these and other FAR proteins in whichsubstitutions are made at residues corresponding to those identifiedherein in the M. algicola FAR protein.

To produce FAR homologs with improved properties, the sequences of thewild-type M. algicola FAR and the FAR homolog (e.g., a M. aquaeolei FARprotein) can be aligned in a pairwise manner as described supra. Basedon the alignment, a residue in a position in the homolog thatcorresponds, based on the alignment, with a specified position in M.algicola FAR is identified. For example, analogous substitutions inwild-type M. aquaeolei FAR to those substitutions described in Table 2can be made by aligning the wild-type M. algicola FAR protein (e.g., SEQID NO:2) with the wild-type M. aquaeolei FAR protein (e.g., SEQ ID NO:5)(see, e.g., FIG. 2). Analogous substitutions in M. aquaeolei FAR aredescribed herein in Example 8.

Thus, in some embodiments, the present invention provides a recombinantFAR variant comprising at least about 70% (or at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99%) sequence identity to SEQ ID NO:2 andcomprising one or more amino acid substitutions selected fromX2D/F/G/H/I/P/N/Q/T/V/W, X3R, X4R, X5S, X6P, X7N, X8K/S, X9D/F, X10T,X12T/V, X14N/R/V/W, X17D, X181, X22E, X24I, X33V, X42L, X50S/V, X54P,X60H, X61R, X62S, X63R/Y, X65G/Q/Y, X69E/Q, X71K, X73K/V, X74K/P,X76K/N/R, X77A/I, X83R, X87G/V, X91I/R, X93V, X98P/R, X101L, X102C,X1041/M, X107C/L/W, X110D, X111S, X112A, X113D/L, X115A/H, X117D, X120V,X121H/S, X122R, X125V, X128H, X132G, X134K/R/S, X137L, X138L/Q, X140C,X142V, X144Q, X148E, X151L, X153I, X160S, X162T, X174C, X177Q/R/T,X180H/R, X185A/I, X186A/G/Y, X188A/I/M/S, X197P, X198Q, X202G, X204G,X205G/P, X207I/L, X209K/N, X212R, X213R, X217L, X220C, X224R, X226A/M,X227A/G/H/R/T, X229R, X236K, X237L, X244A/F/G/H/P, X245N, X246A, X257K,X260R/T, X261D, X263P, X264S, X266A, X269T, X283E/F/K/M/T/V, X287L,X288Q, X2901, X295T/V, X299T, X303G, X3051, X306F/H/N/W, X318I, X328T,X330V, X331V, X332S, X333T, X339G/V, X340P/S/V, X341K, X342L, X350S,X351C, X359L, X364F/I, X365N, X366T/V, X370A/I, X374K/Y, X376P,X377C/K/Y, X380K/N/R, X381C, X384R, X389I/L/M/V, X396G, X397I/L, X398Y,X399T, X400A/L, X401A/C/I/L/S/V, X403C/S, X404A, X405A/C/F/G/L/S/V/W,X406Y, X409V/W/Y, X410A/C/H/N/Q/R/S, X412C/F/M/V, X413L/R, X416L/V,X418I/R/V/Y, X421I/L/N/P/R/S/V/Y, X427K, X429E/K/N/Q/R, X430H/I/R,X432C/Q, X433F/H/K/L/N/W, X436D/K/Q, X437V, X440L, X443T, X444S, X446H,X452A/G/N, X458G/L/M/Q, X459H, X463E/T, X464G, X466E/Q/R, X472V, X474R,X479Q, X484V, X487R/S/T/Y, X490S, X496A, X498A, X499A/H/I/N/P/R/S,X500C/G/H/L/N/P/Q/R/S/W, X501G/R, X502A/P/Q/R/S, X504G/R, X505K,X506G/S, X507A/G/P/R/S, X508D/G/H, X509D/E/G/H/N/R/S/W, X510A/D/G/P/S/Y,X511G/I/K/P/Q/R/S/T, and X512K/S/T, wherein the position is numberedwith reference to a wild-type M. algicola FAR (e.g., SEQ ID NO:2), andwherein the FAR variant exhibits increased fatty alcohol productionrelative to the wild-type FAR homolog from which the FAR variant isderived and/or exhibits an improved fatty alcohol profile relative tothe wild-type FAR from which the FAR variant is derived. In someembodiments, the FAR variant produces an increased amount of fattyalcohols as compared to the wild-type FAR. In some embodiments, the FARvariant produces an increased amount of an aggregate of the fattyalcohols C14:0 (1-tetradecanol), C16:1 (cis Δ⁹-1-hexadecenol), C16:0(1-hexadecanol), C18:1 (cis Δ¹¹-1-octadecenol), and C18:0(1-octadecanol) as compared to the wild-type FAR. In some embodiments,the FAR variant produces a fatty alcohol profile that differs from thefatty alcohol profiles produced by the wild-type FAR. In someembodiments, the FAR variant produces a fatty alcohol profile having anincreased amount of C16:1 (cis Δ⁹-1-hexadecenol) and a decreased amountof C18:1 (cis Δ¹¹-1-octadecenol) as compared to the wild-type FAR. Insome embodiments, the fatty alcohol profile produced by the FAR variantfurther comprises an increased amount of C14:0 (1-tetradecanol) ascompared to the wild-type FAR. In some embodiments, the fatty alcoholprofile produced by the FAR variant further comprises a decreased amountof C16:0 (1-hexadecanol) as compared to the wild-type FAR.

In some embodiments, the present invention relates to a method of makingFAR variants having increased fatty alcohol production and/or animproved fatty alcohol profile relative to wild-type FAR. In someembodiments, the method comprises:

(a) identifying a sequence that comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99%) sequence identity to SEQ ID NO:2(alternatively, SEQ ID NO:5);

(b) aligning the identified sequence with the sequence of SEQ ID NO:2(alternatively, SEQ ID NO:5); and

(c) substituting one or more amino acid residues from the identifiedsequence, wherein the substitutions are made at one or more positionscorresponding to positions selected from A2, T3, Q4, Q5, Q6, Q7, N8, G9,A10, A12, G14, E17, Q18, K22, V24, L33, I42, G50, L54, R60, H61, P62,A63, R65, L69, E71, A73, S74, S76, V77, H83, E87, T91, L93, H98, T101,G102, V104, S107, G110, L111, T112, P113, R115, R117, A120, G121, Q122,A125, N128, S132, N134, E137, E138, D140, A142, K144, L148, E151, V153,N160, A162, N174, N177, Q180, V185, 1186, P188, T197, D198, E202, E204,E205, V207, L209, D212, K213, V217, 8220, K224, L226, E227, K229, R236,E237, S244, D245, T246, L257, K260, A261, S263, G264, S266, I269, S283,1287, E288, V290, A295, A299, E303, V305, S306, V318, 1328, L330, S331,L332, A333, S339, G340, 0341, R342, G350, G351, K359, L364, M365, A366,T370, A374, D376, Q377, Y380, R381, T384, A389, D396, V397, V398, V399,G400, G401, R403, V404, P405, L406, A409, G410, A412, M413, A416, Q418,E421, N427K, D429, T430, R432, S433, T436, 1437, F440, A443, P444, Y446,S452, S458, R459, L463, D464, V466, A472, Q474, L479, 1484, G487, N490,E496, K498, L499, Y500, S501, L502, A504, A505, D506, T507, R508, K509,K510, A511, and A512, wherein the positions are numbered with referenceto SEQ ID NO:2.

In some embodiments, step (c) of the method comprises making one or moreamino acid substitutions selected from A2D/F/G/H/I/P/N/Q/T/V/W, T3R,Q4R, Q5S, Q6P, Q7N, N8K/S, G9D/F, A10T, A12T/V, G14N/R/V/W, E17D, Q18I,K22E, V24I, L33V, 142L, G50S/V, L54P, R60H, H61R, P62S, A63R/Y,R65G/Q/Y, L69E/Q, E71K, A73K/V, S74K/P, S76K/N/R, V77A/I, H83R, E87G/V,T911/R, L93V, H98P/R, T101L, G102C, V104I/M, S107C/L/W, G110D, L111S,T112A, P113D/L, R115A/H, R117D, A120V, G121H/S, Q122R, A125V, N128H,S132G, N134K/R/S, E137L, E138L/Q, D1400, A142V, K144Q, L148E, E151L,V1531, N160S, A162T, N174C, N177Q/R/T, Q180H/R, V185A/I, 1186A/GN,P188A/I/M/S, T197P, D198Q, E202G, E204G, E205G/P, V2071/L, L209K/N,D212R, K213R, V217L, R220C, K224R, L226A/M, E227A/G/H/R/T, K229R, R236K,E237L, S244A/F/G/H/P, D245N, T246A, L257K, K260R/T, A261D, S263P, G264S,S266A, 1269T, S283E/F/K/M/T/V, I287L, E288Q, V290I, A295T/V, A299T,E303G, V3051, S306F/H/N/W, V318I, I328T, L330V, S331V, L332S, A333T,S339G/V, G340P/S/V, Q341K, R342L, G350S, G351C, K359L, L364F/I, M365N,A366T/V, T370A/I, A374K/Y, D376P, Q377C/K/Y, Y380K/N/R, R381C, T384R,A389I/L/M/V, D396G, V397I/L, V398Y, V399T, G400A/L, G401A/C/I/L/S/V,R403C/S, V404A, P405A/C/F/G/L/S/V/W, L406Y, A409V/W/Y,G410A/C/H/N/Q/R/S, A412C/F/MN, M413L/R, A416L/V, Q4181/R/V/Y,E421I/L/N/P/R/S/V/Y, N427K, D429E/K/N/Q/R, T430H/I/R, R432C/Q,S433F/H/K/L/N/W, T436D/K/Q, I437V, F440L, A443T, P444S, Y446H,S452A/G/N, S458G/L/M/Q, R459H, L463E/T, D464G, V466E/Q/R, A472V, Q474R,L479Q, I484V, G487R/S/T/Y, N490S, E496A, K498A, L499A/H/I/N/P/R/S,Y500C/G/H/L/N/P/Q/R/S/W, S501G/R, L502A/P/Q/R/S, A504G/R, A505K,D506G/S, T507A/G/P/R/S, R508D/G/H, K509D/E/G/H/N/R/S/Y, K510A/D/G/P/S/Y,A511G/I/K/P/Q/R/S/T, and A512K/S/T.

In some embodiments, the method further comprises determining whetherthe one or more amino acid substitutions increase fatty alcoholproduction and/or an improved fatty alcohol profile in comparison to thewild-type FAR from which the FAR variant is derived.

M. aquaeolei FAR Variants

In a related aspect FAR variants derived from M. aquaeolei FAR (SEQ IDNO:5) are provided. Tables 4, 4A, and 5 show exemplary substitutions andsubstitution sets. In one aspect, the invention provides a FAR variantcomprising at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity to SEQ ID NO:5 and comprising a substitutionat a substitution position (or set of positions) disclosed in Tables 4,4A, or 5. These variants are sometimes referred to as “FAR Maqvariants.” It will be appreciated, as noted above, that position 512 ofSEQ ID NO:5 corresponds to position 511 of SEQ ID NO:2 and that SEQ IDNO:5 and SEQ ID NO:2 share 78% sequence identity. Accordingly, FAR maqvariants can be viewed as a subgenus of FAR variants of the invention towhich all disclosure herein is applicable.

In some embodiments, a FAR variant comprises at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity to SEQID NO:5 and comprises a substitution relative to SEQ ID NO:5 at one ormore positions selected from position 2, position 135, position 139,position 141, position 422, position 459, position 511, and position512, wherein the position is numbered with reference to SEQ ID NO:5. Insome embodiments, the amino acid residue in the FAR variant at position2 relative to SEQ ID NO:5 is alanine (A2); the amino acid residue atposition 135 relative to SEQ ID NO:5 is asparagine (N135); the aminoacid residue at position 135 relative to SEQ ID NO:5 is glutamic acid(E139); the amino acid residue at position 141 relative to SEQ ID NO:5is aspartic acid (D141); the amino acid residue at position 422 relativeto SEQ ID NO:5 is aspartic acid (D422); the amino acid residue atposition 459 relative to SEQ ID NO:5 is asparagine (N459); the aminoacid residue at position 511 relative to SEQ ID NO:5 is lysine (K511);and the amino acid residue at position 512 relative to SEQ ID NO:5 isalanine (A512). In some embodiments, a FAR variant comprises an aminoacid substitution at residue A2 that is selected from aspartic acid,phenylalanine, glycine, histidine, isoleucine, asparagine, proline,glutamine, threonine, valine, or tryptophan (A2D/F/G/H/I/N/P/Q/T/V/W).In some embodiments, a FAR variant comprises an amino acid substitutionat residue N135 that is selected from lysine, arginine, or serine(N135K/R/S). In some embodiments, a FAR variant comprises an amino acidsubstitution at residue E139 that is selected from leucine or glutamine(E139L/Q). In some embodiments, a FAR variant comprises an amino acidsubstitution at residue D141 that is cysteine (D141C). In someembodiments, a FAR variant comprises an amino acid substitution atresidue D422 that is selected from isoleucine, leucine, asparagine,proline, arginine, serine, valine, or tyrosine (D422I/L/N/P/R/S/V/Y). Insome embodiments, a FAR variant comprises an amino acid substitution atresidue N459 that is selected from glycine, leucine, methionine, orglutamine (N459G/L/M/Q). In some embodiments, a FAR variant comprises anamino acid substitution at residue K511 that is selected from alanine,aspartic acid, glycine, proline, serine, or tyrosine (K511A/D/G/P/S/Y).In some embodiments, a FAR variant comprises an amino acid substitutionat residue A512 that is selected from glycine, isoleucine, lysine,proline, glutamine, arginine, serine, or threonine(A512G/I/K/P/Q/R/S/T). In some embodiments, a FAR variant comprises 100%amino acid sequence identity to SEQ ID NO:5 except for amino acidsubstitutions at one or more of positions A2, N135, E139, D141, D422,N459, K511, and A512 as numbered with reference to SEQ ID NO:5.

In some embodiments, the FAR variant further comprises a substitutionrelative to SEQ ID NO:5 at one or more of position 189, position 406,and position 419, wherein the positions are numbered with reference toSEQ ID NO:5. In some embodiments, the amino acid residue at position 189relative to SEQ ID NO:5 is proline (P189); the amino acid residue atposition 406 relative to SEQ ID NO:5 is proline (P406); and the aminoacid residue at position 419 relative to SEQ ID NO:2 is asparagine(N419). In some embodiments, a FAR variant comprises an amino acidsubstitution at residue P189 that is selected from alanine, isoleucine,methionine, or serine (P189A/I/M/S). In some embodiments, a FAR variantcomprises an amino acid substitution at residue P406 that is selectedfrom alanine, cysteine, phenylalanine, glycine, leucine, serine, valine,or tryptophan (P406A/C/F/G/L/S/V/W). In some embodiments, the FARvariant comprises an amino acid substitution at residue N419 that isselected from isoleucine, arginine, valine, and tyrosine (N419I/R/V/Y).In some embodiments, a FAR variant comprises 100% amino acid sequenceidentity to SEQ ID NO:5 except for amino acid substitutions at one ormore of positions A2, N135, E139, D141, P189, P406, N419, D422, N459,K511, and A512 as numbered with reference to SEQ ID NO:5.

FAR Maq variants of the invention as described herein may have any ofthe improved properties disclosed hereinabove, such as increased totalfatty alcohol production, increased production of fatty alcohols at aspecified culture pH or over an increased pH range, or changes in fattyalcohol profile as compared to a wild-type FAR.

FAR Maq variants of the invention may comprise a substitution orsubstitution set exemplified in Tables 4 or 5. For example, FAR MaqVariant No. 5 (see Table 4) has a threonine (substituted for alanine) atposition 2, and alanine (substituted for glycine) at position 113(numbered with reference to SEQ ID NO:5). More broadly, FAR Maq variantsof the invention may comprise a substitution at a position or set ofpositions exemplified in Table 4 or Table 5. As a non-limiting example,FAR Maq Variant No. 5 (see Table 4) has a substitution for alanine atposition 2 and a substitution for glycine at position 113 (wherein thepositions are numbered with reference to SEQ ID NO:5), and accordingly aparticular FAR Maq variant of the invention may comprise a substitutionat position 2 (i.e., any residue other than alanine) and at position 113(i.e., any residue other than glycine), and optionally any othersubstitutions as described herein, e.g., as described in Table 4 orTable 5.

Increased Thermotolerance

As described in the examples, screening of some FAR variants includedculture at elevated temperature (e.g., 37° C. or 40° C., rather than 30°C.). See, e.g., Table 2. In these experiments a number of substitutionswere identified that may increase the thermotolerance of a FARpolypeptide (i.e., allow the polypeptide to retain activity at elevatedtemperatures). Alternatively, these substitutions may shift thetemperature optimum of the variant from a lower temperature to a highertemperature.

For example, the following substitutions to variant 391 resulted inincreased activity at 37° C.: G14R/V, V104I/M, S134R, E227R, S244P,S283M, S306W, L364I, T370I, D376P, Q377K, A389I, and S433K, numberedwith reference to SEQ ID NO:2. It is believed that introducingsubstitutions into one or more of positions G14, V104, S134, E227, S244,S283, S306, L364, T370, D376, Q377, A389, and S433, numbered withreference to SEQ ID NO:2, into any of Variants 1-629 of Table 2, or intocorresponding positions in any of Variants 1-629 of Table 5, will resultin improved activity when the host cell (e.g., E. coli) is incubated atan elevated temperature (e.g., 37° C.). For example, it is believed thatintroducing one or more of the following substitutions into any ofVariants 1-629 of Table 2, or equivalent substitutions into any ofVariants 1-629 of Table 5, will result in improved activity when thehost cell (e.g., E. coli) is incubated at an elevated temperature (e.g.,≧37° C.): G14R/V, V104I/M, S134R, E227R, S244P, S283M, S306W, L364I,T370I, D376P, Q377K, A389I, or S433K.

For example, the following substitution sets to variant 438 resulted inincreased activity at 40° C.: Q18I, A63R, R65G, N128H, S134R, N174C,N177T, K224R, L226M, S283F, G351C, M365N, V404A, L406Y, K433S, andG487R, numbered with reference to SEQ ID NO:2. It is believed thatintroducing substitutions into one or more of positions Q18, A63, R65,N128, S134, N174, N177, K224, L226, S283, G351, M365, V404, L406, K433,or G487, numbered with reference to SEQ ID NO:2 into any of Variants1-629 of Table 2, or into corresponding positions in any of Variants1-629 of Table 5, will result in improved activity when the host cell(e.g., E. coli) is incubated at an elevated temperature (e.g., 40° C.).For example, it is believed that introducing one or more of thefollowing substitutions into any of Variants 1-629 of Table 2, orequivalent substitutions into any of Variants 1-629 of Table 5, willresult in improved activity when the host cell (e.g., E. coli) isincubated at an elevated temperature (e.g., 40° C.): Q18I, A63R, R65G,N128H, S134R, N174C, N177T, K224R, L226M, S283F, G351C, M365N, V404A,L406Y, K433S, or G487R.

Generation of FAR Variants

A FAR variant of the present invention can be subject to furthermodification to generate new polypeptides that retain the specificsubstitutions that characterize the variant and which may have desirableproperties. For example, a polynucleotide encoding a FAR with animproved property can be subjected to additional rounds of mutagenesisto generate polypeptides that retain the properties of the parent orexhibit further improvements in the desired enzyme or enzyme properties.

Methods for generating variant libraries of polynucleotides encodingmodified polypeptides are well known in the art. For example,mutagenesis and directed evolution methods can be readily applied topolynucleotides encoding the FAR polypeptide of SEQ ID NO: 2 to generatevariant libraries that can be expressed, screened, and assayed using themethods described herein: Mutagenesis and directed evolution methods arewell known in the art. See, e.g., Ling, et al., “Approaches to DNAmutagenesis: an overview,” Anal. Biochem., 254(2):157-78 (1997); Dale,et al., “Oligonucleotide-directed random mutagenesis using thephosphorothioate method,” Methods Mol. Biol., 57:369-74 (1996); Smith,“In vitro mutagenesis,” Ann. Rev. Genet., 19:423-462 (1985); Botstein,et al., “Strategies and applications of in vitro mutagenesis,” Science,229:1193-1201 (1985); Carter, “Site-directed mutagenesis,” Biochem. J.,237:1-7 (1986); Kramer, et al., “Point Mismatch Repair,” Cell,38:879-887 (1984); Wells, et al., “Cassette mutagenesis: an efficientmethod for generation of multiple mutations at defined sites,” Gene,34:315-323 (1985); Minshull, et al., “Protein evolution by molecularbreeding,” Current Opinion in Chemical Biology, 3:284-290 (1999);Christians, et al., “Directed evolution of thymidine kinase for AZTphosphorylation using DNA family shuffling,” Nature Biotechnology,17:259-264 (1999); Crameri, et al., “DNA shuffling of a family of genesfrom diverse species accelerates directed evolution,” Nature,391:288-291; Crameri, et al., “Molecular evolution of an arsenatedetoxification pathway by DNA shuffling,” Nature Biotechnology,15:436-438 (1997); Zhang, et al., “Directed evolution of an effectivefucosidase from a galactosidase by DNA shuffling and screening,”Proceedings of the National Academy of Sciences, U.S.A., 94:45-4-4509;Crameri, et al., “Improved green fluorescent protein by molecularevolution using DNA shuffling,” Nature Biotechnology, 14:315-319 (1996);Stemmer, “Rapid evolution of a protein in vitro by DNA shuffling,”Nature, 370:389-391 (1994); Stemmer, “DNA shuffling by randomfragmentation and reassembly: In vitro recombination,” Proceedings ofthe National Academy of Sciences, U.S.A., 91:10747-10751 (1994); WO95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO01/75767; and WO 2009/152336, all of which are incorporated herein byreference.

V. Polynucleotides and Expression Systems Encoding Far Variants

In another aspect, the present invention provides polynucleotidesencoding the FAR variants as described herein. The polynucleotide can bea DNA or RNA, and can be single-stranded or double-stranded. Thepolynucleotide may be operably linked to one or more heterologousregulatory or control sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide.Expression constructs containing a heterologous polynucleotide encodingthe engineered FAR variant can be introduced into appropriate host cellsto express the FAR variant.

In some embodiments, the FAR variant is generated from a wild-type FARcDNA sequence (e.g., a wild-type M. algicola FAR cDNA sequence of SEQ IDNO:1 or SEQ ID NO:3 or a wild-type M. aquaeolei FAR cDNA sequence of SEQID NO:4) or the portion thereof comprising the open reading frame, withchanges made as required at the codons corresponding to substitutions(residues mutated relative to the wild-type sequence as describedherein, for example at any of Tables 2, 4, or 5). In addition, one ormore “silent” nucleotide changes can be incorporated. These changes mayaffect cellobiohydrolase activity in a variety of ways. For example,without intending to be bound by a particular mechanism, silentmutations may increase the stability of mRNAs encoding the variantprotein.

The availability of a polypeptide sequence of a specific improved FARpolypeptide provides a description of all polynucleotides capable ofencoding that enzyme or fragment because of the known correspondencebetween amino acids and the genetic code. For most orgamisms the geneticcode is “Amino Acid (one letter code) [codons]”: phenylalanine (F) [TTT,TTC]; leucine (L) [TTA, TTG, CTT, CTC, CTA, CTG]; isoleucine (I) [ATT,ATC, ATA]; methionine (M) [ATG]; valine (V) [TGG, GTC, GTA, GTG]; serine(S) [TCT, TCC, TCA, TCG, AGT, AGC]; proline (P) [CCT, CCC, CCA, CCG];threonine (T) [ACT, ACC, ACA, ACG]; alanine (A) [GCT, GCC, GCA, GCG];tyrosine (Y) [TAT, TAC]; histidine (H) [CAT, CAC]; glutamine (Q) [CAA,CAG]; asparagine (N) [AAT, AAC]; lysine (K) AAA, AAG]; aspartic acid (D)[GAT, GAC]; glutamic acid (E) [GAA, GAG]; cysteine (C) [TGT, TGC];tryptophan (W) [TGG]; arginine (R) [CGT, CGC, CGA, CGG, AGA, AGG]; andglycine (G) [GGT, GGC, GGA, GGG]. In certain embodiments, the degeneracyof the genetic code is used to produce a large number of polynucleotidesthat encode the improved FAR polypeptides described herein. In someembodiments, the polynucleotides that encode the improved FARpolypeptides described herein are codon optimized for expression inspecific microorganisms. In particular embodiments, the polynucleotidesthat encode the improved FAR polypeptides described herein are codonoptimized for expression in bacteria, yeast or filamentous fungi. Insome embodiments, the polynucleotides are codon optimized for expressionin oleaginous yeast. In other specific embodiments, the polynucleotidesare codon optimized for expression in E. coli, S. cerevisiae or Y.lipolytica. Codon schemes and/or methods for determining codon schemesoptimized for particular microorganisms of interest are well known (see,e.g., the references cited with the definition of “preferred, optimalhigh, codon usage bias codons”; see also the websitewww.kazusa.or.jp/codon/).

A variety of methods are known for determining the codon frequency(e.g., codon usage, relative synonymous codon usage) and codonpreference in specific organisms, including multivariate analysis, forexample, using cluster analysis or correspondence analysis, and theeffective number of codons used in a gene (see GCG CodonPreference,Genetics Computer Group Wisconsin Package; Codon W, John Peden,University of Nottingham; McInerney, J. O, 1998, Bioinformatics14:372-73; Stenico et al., 1994, Nucleic Acids Res. 222437-46; Wright,F., 1990, Gene 87:23-29; Wada et al., 1992, Nucleic Acids Res.20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res. 28:292; Henaut andDanchin, “Escherichia coli and Salmonella,” 1996, Neidhardt, et al.Eds., ASM Press, Washington D.C., p. 2047-2066, all of which areincorporated herein be reference). The data source for obtaining codonusage may rely on any available nucleotide sequence capable of codingfor a protein. These data sets include nucleic acid sequences actuallyknown to encode expressed proteins (e.g., complete protein codingsequences-CDS), expressed sequence tags (ESTs), or predicted codingregions of genomic sequences (see for example, Mount, D.,Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E.C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput.Appl. Biosci. 13:263-270, all of which are incorporated herein byreference).

In some embodiments, the present invention provides a method for makingan improved FAR polynucleotide variant, wherein the method comprisesintroducing one or more mutations into a polynucleotide encoding a FARwhich comprises at least 70% (at least 75%, at least 80%, at least 85%,at least 90%, at least 93%, or at least 95%) sequence identity to theamino acid sequence of SEQ ID NO:2 or a functional fragment thereof toproduce a modified polynucleotide, wherein the modification is selectedfrom the group consisting of a substitution, a deletion, and aninsertion; transforming a host cell with the modified polynucleotide;and screening the transformed host cell for an improvement in a desiredphenotype relative to a corresponding transformed host cell comprising apolynucleotide encoding a wild-type FAR having at least 70% (at least75%, at least 80%, at least 85%, at least 90%, at least 93%, at least95%) sequence identity to the amino acid sequence of SEQ ID NO:2 or afunctional fragment thereof. Exemplary phenotypes include improved fattyalcohol production, total and/or secreted fatty alcohol composition,and/or alteration of the fatty alcohol composition (including, but notlimited to, an increase in the amount of C18 fatty alcohols compared tothe amount of C14 fatty alcohols comprising the composition, anincreased amount of C14-C16 fatty alcohols produced, or a profilecomprising one or more of an increased amount of C16:1 (cisΔ⁹-1-hexadecenol), and a decreased amount of C18:1 (cisΔ¹¹-1-octadecenol), an increased amount of C14:0 (1-tetradecanol), and adecreased amount of C16:0 (1-hexadecanol) as compared to wild-type FAR).Accordingly, in some embodiments the invention relates to apolynucleotide sequence that encodes an improved FAR polypeptide whereinthe polynucleotide is selected from the group consisting of apolynucleotide that encodes a fatty acyl reductase (FAR) polypeptidecomprising a sequence that is at least 75%, at least 80%, at least 85%,at least 90%, at least 95% or at least 97% identical to SEQ ID NO:2 andsaid improved FAR polypeptide comprising one or more substitutions. Insome embodiments, the one or more substitutions comprise an amino acidsubstitution set listed in Table 2, Table 4, or Table 5. In someembodiments, the one or more substitutions comprise a substitution atany one of positions 2, 134, 138, 188, 405, 511 or combinations thereofwhen aligned to SEQ ID NO:2.

In some embodiments, the polynucleotide encodes a full-length improvedFAR polypeptide wherein the corresponding full-length FAR enzyme has atleast 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99%) sequence identity with the wild-type FAR ofSEQ ID NO:2 and also includes one or more of the specified substitutionsas described herein (see, e.g., Section II supra). In some embodiments,the improved FAR comprises an amino acid sequence encoded by a nucleicacid that hybridizes under stringent conditions over substantially theentire length of a nucleic acid corresponding to SEQ ID NO:1, SEQ IDNO:3, or SEQ ID NO:4.

In some embodiments, the polynucleotide encoding an improved FARpolypeptide will have at least 90%, at least 93%, at least 94%, at least95%, at least 96%, at least 97% or at least 98% sequence identity tonucleic acid sequences of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:4.

Certain silent mutations have also been identified in polynucleotidesencoding the improved FAR polypeptides which appear to confer theproperty of increased fatty alcohol production in transformed E. colicells as compared to wild-type M. algicola DG893 FAR (see Table 2). Thesilent mutations include: t6a, t6g, t6c, t9c, t9g, t27c, a30g, a51g,a81t, a147t, c171t, t174c, t180c, t226a, a237g, g243a, a318g, c321g,c321a, a336c, t339g, t363c, c402t, t459c, a474g, a540g, t564g, a615g,g627t, t628c, a633g, g681a, g711a, t792c, t834c, t870c, t927c, t967c,c994t, t1026c, t1149c, c1173t, t1203c, t1236g, t1248c, g1263a, g1272a,c1281t, t1287c, g1290c, t1297a, c1299g, t1326c, t1357c, c1366t, t1372a,t1374g, t1398c, t1410c, t1413c, t1435c, t1461g, g1485a, g1497t, t1501a,t1504c, t1515g, t1515a, t1521c, t1524c, a1527g, and t1533c (wherenucleotide position is determined by alignment with SEQ ID NO:1).

Polynucleotide Synthesis

Polynucleotides encoding FAR polypeptides can be prepared using methodsthat are well known in the art. Typically, oligonucleotides of up toabout 40 bases are individually synthesized, then joined (e.g., byenzymatic or chemical ligation methods, or polymerase-mediated methods)to form essentially any desired continuous sequence. For example,polynucleotides of the present invention can be prepared by chemicalsynthesis using, for example, the classical phosphoramidite methoddescribed by Beaucage, et al., 1981, Tetrahedron Letters, 22:1859-69, orthe method described by Matthes, et al., 1984, EMBO J. 3:801-05, both ofwhich are incorporated herein by reference. These methods are typicallypracticed in automated synthetic methods. According to thephosphoramidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

In addition, essentially any nucleic acid can be custom ordered from anyof a variety of commercial sources, such as The Midland CertifiedReagent Company (Midland, Tex.), The Great American Gene Company(Ramona, Calif.), ExpressGen Inc. (Chicago, Ill.), Operon TechnologiesInc. (Alameda, Calif.), and many others.

Polynucleotides may also be synthesized by well-known techniques asdescribed in the technical literature. See, e.g., Carruthers, et al.,1982, Cold Spring Harbor Symp. Quant. Biol., 47:411-18 and Adams et al.,1983, J. Am. Chem. Soc. 105:661, both of which are incorporated hereinby reference. Double stranded DNA fragments may then be obtained eitherby synthesizing the complementary strand and annealing the strandstogether under appropriate conditions, or by adding the complementarystrand using DNA polymerase with an appropriate primer sequence.

General texts that describe molecular biological techniques which areuseful herein, including the use of vectors, promoters, protocolssufficient to direct persons of skill through in vitro amplificationmethods, including the polymerase chain reaction (PCR) and the ligasechain reaction (LCR), and many other relevant methods, include Bergerand Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymologyvolume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook etal., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”)and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (supplemented through 2009)(“Ausubel”), all of which are incorporated herein by reference.Reference is made to Berger, Sambrook, and Ausubel, as well as Mullis etal., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methodsand Applications (Innis et al. eds) Academic Press Inc. San Diego,Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; TheJournal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl.Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci.USA 87, 1874; Lomeli et al. (1989) J. Clin. Chem 35, 1826; Landegren etal., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8,291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990)Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564,all of which are incorporated herein by reference. Methods for cloningin vitro amplified nucleic acids are described in Wallace et al., U.S.Pat. No. 5,426,039, which is incorporated herein by reference.

Vectors

The present invention further provides DNA constructs and vectorscomprising polynucleotides encoding the improved FAR polypeptides forexpression in heterologous recombinant host cells. In certainembodiments, the DNA constructs and vectors comprise a polynucleotidesequence that encodes any one of the improved FAR polypeptides asdisclosed above. In certain embodiments, the DNA constructs and vectorscomprise a polynucleotide sequence as encompassed by the invention anddisclosed herein above. In certain embodiments, the DNA constructs andvectors comprise a polynucleotide sequence that encodes an improved FARpolypeptide herein, wherein the improved FAR is a full-length FAR. Inother embodiments, the DNA constructs and vectors comprise apolynucleotide sequence that encodes an improved FAR polypeptide,wherein the improved FAR is a functional fragment of an improvedfull-length FAR enzyme. In certain embodiments, the polynucleotidesencoding improved FAR polypeptides for expression in heterologousrecombinant host cells as described herein are operably linked to apromoter, and optionally, to other control sequences.

In a particular aspect the present invention provides an expressionvector comprising a FAR polynucleotide operably linked to a heterologouspromoter. Expression vectors of the present invention may be used totransform an appropriate host cell to permit the host to express the FARprotein. Methods for recombinant expression of proteins in bacteria,yeast, and other organisms are well known in the art, and a numberexpression vectors are available or can be constructed using routinemethods.

A recombinant expression vector can be any vector, e.g., a plasmid or avirus, which can be manipulated by recombinant DNA techniques tofacilitate expression of an improved FAR polypeptide in a recombinanthost cell. In certain embodiments, the expression vectors is stablyintegrated into the chromosome of the recombinant host cell andcomprises one or more heterologous genes operably linked to one or morecontrol sequences useful for production of an improved FAR polypeptide.In other embodiments, the expression vector is an extrachromosomalreplicative DNA molecule, e.g., a linear or closed circular plasmid,that is found either in low copy number (e.g., from about 1 to about 10copies per genome equivalent) or in high copy number (e.g., more thanabout 10 copies per genome equivalent).

Expression vectors which, in certain embodiments, are useful forexpressing improved FAR enzymes as disclosed herein are commerciallyavailable, e.g., from Sigma-Aldrich Chemicals, St. Louis, Mo. andStratagene, LaJolla, Calif. In some embodiments, examples of suitableexpression vectors are plasmids which are derived from pBR322 (GibcoBRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (Lathe et al.,1987, Gene 57:193-201).

In certain embodiments, the present disclosure provides a plasmid forexpression of heterologous genes in E. coll. Expression vectorpCK11900,which comprises a P15A origin of replication (P15A ori), lac a CAPbinding site, a lac promoter, a T7 ribosomal binding site (T7g10 RBS)and a chloramphenicol resistance gene (camR). This expression vector isdepicted in FIG. 3 of U.S. Patent Publication No. 2006/0195947, which isincorporated herein by reference in its entirety.

In certain embodiments, the present disclosure provides a replicatingplasmid for expression of heterologous genes in Yarrowia, andparticularly in Y. lipolytica.

In some embodiments, expression vectors as described herein are adaptedfor overexpression of genes encoding enzymes other than improved FARpolypeptides that are directly involved in fatty acid biosynthesis. Inparticular embodiments, the overexpressed gene encodes a proteinselected from a fatty acid synthase (FAS), an acyl-ACP thioesterase(TE), a fatty acyl-CoA synthase (FACS) and an acetyl-CoA carboxylase(ACC). In some embodiments, the expression vector encoding the improvedFAR enzyme and the expression vector encoding a second enzyme (e.g., anFAS, TE, FACS or ACC) are separate nucleic acids. In other embodiments,the improved FAR enzyme and the second enzyme are encoded on the sameexpression vector, and expression of each enzyme is independentlyregulated by a different promoter.

In various embodiments, an expression vector optionally contains aribosome binding site (RBS) for translation initiation, and atranscription terminator, such as PinII. The vector also optionallyincludes appropriate sequences for amplifying expression, e.g., anenhancer.

Promoters

Suitable promoters include constitutive promoters, regulated promoters,and inducible promoters. Appropriate promoter sequences can be obtainedfrom genes encoding extracellular or intracellular polypeptides whichare either endogenous or heterologous to the host cell. Methods for theisolation, identification and manipulation of promoters of varyingstrengths are available in or readily adapted from the art. See, e.g.,Nevoigt et al. (2006) Appl. Environ. Microbiol. 72:5266-5273, thedisclosure of which is herein incorporated by reference in its entirety.

In certain embodiments, the DNA constructs and vectors comprising apolynucleotide encoding an improved FAR polypeptides are suitable forexpression in bacteria. For bacterial host cells, suitable promoters fordirecting transcription of the nucleic acid constructs of the presentdisclosure, include the promoters obtained from the E. coli lac operon,Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, Bacillus megaterium promoters, and prokaryotic beta-lactamasegene (Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75:3727-3731(1978)), as well as the tac promoter (DeBoer et al., Proc. NatlAcad. Sci. USA 80: 21-25(1993)). Additional promoters include trppromoter, phage lambda PL, T7 promoter, promoters found at PromEC andthe like. Promoters suitable for use in the present disclosure aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican 242:74-94 (1980); and in Sambrook et al (2001) MolecularCloning: A Laboratory Manual, 3^(rd) ed., Cold Spring Harbor LaboratoryPress, New York.

In various embodiments, the DNA constructs and vectors comprisingpolynucleotides encoding an improved FAR polypeptide are suitable forexpression in yeast. In certain embodiments, the DNA constructs andvectors comprising the polynucleotides encoding an improved FAR aresuitable for expression in oleaginous yeast, such as but not limited toYarrow lipolytica. In certain embodiments the promoter is a Y.lipolytica promoter.

In certain embodiments, the DNA constructs and vectors comprising thepolynucleotides encoding an improved FAR polypeptide are suitable forexpression in yeast, such as but not limited to S. cerevisiae. For yeasthost cells, suitable promoters for directing transcription of thenucleic acid constructs of the present disclosure are known to theskilled artisan and include, but are not limited to, an enolase(ENO-1_gene) promoter, a galactokinase (GAL1) promoter, an alcoholdehyrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP)promoter, a translation elongation factor EF-1 alpha (TEF1) promoter aswell as those described by Romanos et al. (1992) Yeast 8:423-488. Inother embodiments, promoters include the TEF1 promoter and an RPS7promoter.

In various embodiments, the DNA constructs and vectors comprisingpolynucleotides encoding an improved FAR polypeptide are suitable forexpression in filamentous fungal host cells. For these cells, suitablepromoters for directing the transcription of the nucleic acid constructsof the present disclosure include promoters obtained from the genes forAspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase,Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, and Fusarium oxysporum trypsin-like protease (WO 96/00787),as well as the NA2-tpi promoter (a hybrid of the promoters from thegenes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase), and mutant, truncated, and hybrid promotersthereof. Examples of suitable promoters useful for directing thetranscription of the nucleotide constructs of the present invention in afilamentous fungal host cell are promoters such as cbh1, cbh2, egl1,egl2, pepA, hfb1, hfb2, xyn1, amy, and glaA (Nunberg et al., Mol. CellBiol., 4:2306-2315 (1984), Boel et al., EMBO J 3:1581-1585 ((1984) andEPA 137280).

Other Regulatory Elements

In various embodiments, the polynucleotides useful for expressingheterologous FAR enzymes in recombinant host cells are operably linkedto other control sequences, including but not limited to, atranscription terminator sequence, a signal sequence that whentranslated directs the expressed polypeptide into the secretory pathwayof the recombinant host cell, and a polyadenylation sequence(eukaryotes). The choice of appropriate control sequences for use in thepolynucleotide constructs of the present disclosure is within the skillin the art and in various embodiments is dependent on the recombinanthost cell used and the desired method of recovering the fatty alcoholcompositions produced.

In various embodiments, the expression vector includes one or moreselectable markers, which permit easy selection of transformed cells.Selectable markers for use in a host organism as described hereininclude, but are not limited to, genes that confers antibioticresistance (e.g., ampicillin, kanamycin, chloramphenicol or tetracyclineresistance) to the recombinant host organism that comprises the vector.

VI. Host Cells Comprising Far Variants

In some embodiments, the present invention provides a method forproducing a recombinant host cell, wherein the method comprises: (a)providing a nucleic acid construct of the present invention, wherein thenucleic acid construct comprises polynucleotide encoding an improved FARpolypeptide as described herein; and (b) transforming a host cell withthe nucleic acid construct to produce a recombinant cell. In someembodiments, the host cell is a bacterial cell. In some embodiments, thehost cell is a yeast cell. The transformed or transfected host cell iscultured in a suitable nutrient medium under conditions permitting theexpression of the FAR enzyme. The medium used to culture the cells maybe any conventional medium suitable for growing the host cells, such asminimal or complex media containing appropriate supplements. Suitablemedia are available from commercial suppliers or may be preparedaccording to published recipes (e.g. in catalogues of the American TypeCulture Collection).

Host Cells

The recombinant host cells or microorganisms of the present inventiongenerally comprise a polynucleotide, such as one of the polynucleotidesdescribed above, encoding an improved FAR polypeptide. Suitable hostmicroorganisms include, but are not limited to, bacteria, yeast,filamentous fungi and algae. In certain embodiments, the yeast is anoleaginous yeast. In certain embodiments, microorganisms useful asrecombinant host cells are wild-type microorganisms.

In various embodiments, microorganisms useful as recombinant host cellsare genetically modified. As used herein, “genetically modified”microorganisms include microorganisms having one or more endogenousgenes removed, microorganisms having one or more endogenous genes withreduced expression compared to the parent or wild-type microorganism, ormicroorganisms having one or more genes overexpressed compared to theparent or wild-type microorganism. In certain embodiments, the one ormore genes that are overexpressed are endogenous to the microorganism.In some embodiments, the one or more genes that are overexpressed areheterologous to the microorganism.

In certain embodiments, the genetically modified microorganism comprisesan inactivated or silenced endogenous gene that codes for a proteininvolved in the biosynthesis of fatty acyl-CoA substrates. In particularembodiments, the inactive or silenced gene encodes a fatty acyl-ACPthioesterase or a fatty acyl-CoA synthetase (FACS).

In certain embodiments, the genetically modified microorganismoverexpresses a gene that encodes one or more proteins other than animproved FAR enzyme. In various embodiments, the one or moreoverexpressed proteins increase the rate at which the recombinant cellproduces the acyl-thioester FAR substrate, e.g., the compound of formula(I) shown above. In some embodiments, the one or more overexpressedgenes encodes a protein directly involved in fatty acid biosynthesis. Inparticular embodiments, the one or more overexpressed genes encode aprotein selected from a fatty acid synthase (FAS), an acyl-ACPthioesterase (TE), a fatty acyl-CoA synthase (FACS) and an acetyl-CoAcarboxylase (ACC). In some embodiments, the overexpressed gene isendogenous to the microorganism. In other embodiments, the overexpressedgene is heterologous to the microorganism.

Prokaryotic Host Cells

In some embodiments, the host cell is a prokaryotic cell. Suitableprokaryotic cells include gram positive, gram negative and gram-variablebacterial cells. In certain embodiments, host cells include, but are notlimited to, species of a genus selected from the group consisting ofAgrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter,Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium,Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter,Clostridium, Corynebacterium, Chromatium, Coprococcus, Cyanobacteria,Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium,Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus,Helicobacter, Klebsiella, Lactobacillus, Lactococcus, llyobacter,Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium,Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas,Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas,Roseburia, Rhodospirillum, Rhodococcus, Scenedesmun, Streptomyces,Streptococcus, Synnecoccus, Saccharomonospora, Staphylococcus, Serratia,Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis,Temecula, The rmosynechococcus, Thermococcus, Ureaplasma, Xanthomonas,Xylella, Yersinia and Zymomonas. In particular embodiments, the hostcell is a species of a genus selected from the group consisting ofAgrobacterium, Arthrobacter, Bacillus, Clostridium, Corynebacterium,Escherichia, Erwinia, Geobacillus, Klebsiella, Lactobacillus,Mycobacterium, Pantoea, Rhodococcus, Streptomyces and Zymomonas.

In certain embodiments, the recombinant host cell is an industrialbacterial strain. Numerous bacterial industrial strains are known andsuitable for use in the methods disclosed herein. In some embodiments,the bacterial host cell is a species of the genus Bacillus, e.g., B.thuringiensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B.circulars, B. pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B.alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B.halodurans and B. amyloliquefaciens. In particular embodiments, the hostcell is a species of the genus Bacillus and is selected from the groupconsisting of B. subtilis, B. pumilus, B. licheniformis, B. clausii, B.stearothermophilus, B. megaterium and B. amyloliquefaciens.

In some embodiments the bacterial host cell is a species of the genusErwinia, e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E.punctata or E. terreus.

In other embodiments the bacterial host cell is a species of the genusPantoea, e.g., P. citrea or P. agglomerans.

In still other embodiments, the bacterial host cell is a species of thegenus Streptomyces, e.g., S. ambofaciens, S. achromogenes, S.avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus,S. griseus or S. lividans.

In further embodiments, the bacterial host cell is a species of thegenus Zymomonas, e.g., Z. mobilis or Z lipolytica.

In further embodiments, the bacterial host cell is a species of thegenus Rhodococcus, e.g. R. opacus.

In particular embodiments, the bacterial host cell is a species of thegenus Escherichia, e.g., E. coli. In certain embodiments, the E. coli isa wild-type bacterium. In various embodiments, the wild-type E. colibacterial strain useful in the processes described herein is selectedfrom, but not limited to, strain W3110, strain MG1655 and strainBW25113. In other embodiments, the E. coli is genetically modified.Examples of genetically modified E. coli useful as recombinant hostcells include, but are not limited to, genetically modified E. colifound in the Keio Collection, available from the National BioResourceProject at NBRP E. coli, Microbial Genetics Laboratory, NationalInstitute of Genetics 1111 Yata, Mishima, Shizuoka, 411-8540.

In particular embodiments, the genetically modified E. coli comprises aninactivated or silenced endogenous fadD gene, which codes for anacyl-CoA synthetase protein. In other embodiments the geneticallymodified E. coli comprises an inactivated of silenced endogenous fadKgene, which codes for an endogenous short-chain acyl-CoA synthetase. Instill other embodiments, the genetically modified E. coli comprises aninactivated or silenced endogenous fadD gene and an inactivated orsilenced endogenous fadK gene. In other embodiments, the geneticallymodified E. coli comprises an endogenous fadD gene that has reducedexpression compared to the parent or wild-type strain. In variousembodiments, the genetically modified E. coli comprises an endogenousfadK gene that has reduced expression compared to the parent orwild-type strain.

Yeast Host Cells

In certain embodiments, the recombinant host cell is a yeast. In variousembodiments, the yeast host cell is a species of a genus selected fromthe group consisting of Candida, Hansenula, Saccharomyces,Schizosaccharomyces, Pichia, Kluyveromyces, and Yarrowia. In particularembodiments, the yeast host cell is a species of a genus selected fromthe group consisting of Saccharomyces, Candida, Pichia and Yarrowia.

In various embodiments, the yeast host cell is selected from the groupconsisting of Hansenula polymorpha, Saccharomyces cerevisiae,Saccaromyces carlsbergensis, Saccharomyces diastaticus, Saccharomycesnorbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichiapastoris, Pichia finlandica, Pichia trehalophila, Pichia ferniemtans,lssatchenkia orientalis, Pichia kodamae, Pichia membranaefaciens, Pichiaopuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum,Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta,Kluyveromyces lactis, Candida albicans, Candida krusei, Candidaethanolic and Yarrowia lipolytica and synonyms or taxonomic equivalentsthereof.

In certain embodiments, the yeast host cell is a wild-type cell. Invarious embodiments, the wild-type yeast cell strain is selected from,but not limited to, strain BY4741, strain FL100a, strain INVSC1, strainNRRL Y-390, strain NRRL Y-1438, strain NRRL YB- 1952, strain NRRLY-5997, strain NRRL Y-7567, strain NRRL Y-1532, strain NRRL YB-4149andstrain NRRL Y-567. In other embodiments, the yeast host cell isgenetically modified. Examples of genetically modified yeast useful asrecombinant host cells include, but are not limited to, geneticallymodified yeast found in the Open Biosystems collection found at thewebsite www.openbiosystems.com/GeneExpression/Yeast/YKO/. See Winzeleret al. (1999) Science 285:901-906.

In other embodiments, the recombinant host cell is an oleaginous yeast.Oleaginous yeast are organisms that accumulate lipids such astri-acylglycerols. Examples of oleaginous yeast include, but are notlimited to, organisms selected from the group consisting of Yarrowialipolytica, Yarrowia paralipolytica, Candida revkaufi, Candidapulcherrima, Candida tropicalis, Candida utilis, Candida curvata D,Candida curvata R, Candida diddensiae, Candida boldinii, Rhodotorulaglutinous, Rhodotorula graminis, Rhodotorula mucilaginosa, Rhodotorulaminuta, Rhodotorula bacarum, Rhodosporidium toruloides, Cryptococcus(terricolus) albidus var. albidus, Cryptococcus laurentii, Trichosporonpullans, Trichosporon cutaneum, Trichosporon cutancum, Trichosporonpullulans, Lipomyces starkeyii, Lipomyces lipoferus, Lipomycestetrasporus, Endomycopsis vernalis, Hansenula ciferri, Hansenulasaturnus, and Trigonopsis variables. In particular embodiments, theoleaginous yeast is Y. lipolytica. In certain embodiments, Yarrowialipolytica strains include, but are not limited to, DSMZ 1345, DSMZ3286, DSMZ 8218, DSMZ 70561, DSMZ 70562, and DSMZ 21175.

In certain embodiments, the oleaginous yeast is a wild-type organism. Inother embodiments, the oleaginous yeast is genetically modified.

In yet other embodiments, the recombinant host cell is a filamentousfungus. In certain embodiments, the filamentous fungal host cell is aspecies of a genus selected from the group consisting of Achlya,Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria,Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium,Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor,Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia,Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum,Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium,Trichoderma, Verticillium, Volvariella, and teleomorphs, synonyms ortaxonomic equivalents thereof.

In some embodiments, the filamentous fungal host cell is an Aspergillusspecies, a Chrysosporium species, a Corynascus species, a Fusariumspecies, a Humicola species, a Myceliophthora species, a Neurosporaspecies, a Penicillum species, a Tolypocladium species, a Tramatesspecies, or Trichoderma species. In other embodiments, the Trichodermaspecies is selected from T. longibrachiatum, T. viride, Hypocreajecorina and T. reesei; the Aspergillus species is selected from A.awamori, A. funigatus, A. japonicus, A. nidulans, A. niger, A.aculeatus, A. foetidus, A. oryzae, A. sojae, and A. kawachi; theChrysosporium species is C. lucknowense; the Fusarium species isselected from F. graminum, F. oxysporum and F. venenatum; theMyceliophthora species is M. thermophilia; the Neurospora species is N.crassa; the Humicola species is selected from H. insolens, H. grisea,and H. lanuginosa; the Penicillum species is selected from P.purpurogenum, P. chtysogenum, and P. verruculosum; the Thielavia speciesis T. terrestris; and the Trametes species is selected from T. villosaand T. versicolor.

In some embodiments, the filamentous fungal host is a wild-typeorganism. In other embodiments, the filamentous fungal host isgenetically modified.

In certain particular embodiments, recombinant host cells for use in themethods described herein are derived from strains of Escherichia coli,Bacillus, Saccharomyces, Streptomyces and Yarrowia.

In certain embodiments the host cell is a Yarrowia cell, such as a Y.lipolytica cell.

Cells which are useful in the practice of the present disclosure includeprokaryotic and eukaryotic cells which are readily accessible from anumber of culture collections and other sources, e.g., the American TypeCulture Collection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ) (German Collection of Microorganisms and CellCulture), Centraalbureau Voor Schimmelcultures (CBS), and AgriculturalResearch Service Patent Culture Collection, Northern Regional ResearchCenter (NRRL). Yarrowia lipolytica is available, as a non-limitingexample, from the ATCC under accession numbers 20362, 18944, and 76982.

In some embodiments, the method of producing fatty alcohols may beachieved in a recombinant microorganism by one or two differentbiosynthetic pathways. These pathways are described as an acyl-CoAindependent pathway or an acyl-CoA dependent pathway and reference ismade to FIG. 1. Accordingly fatty alcohols can be produced by arecombinant microorganism that lacks a functional endogenous enzymeinvolved in the biosynthesis of fatty acyl-CoA substrates. Thus in someaspects, fatty alcohols can be produced by a recombinant microorganismthat express a gene encoding a improved FAR polypeptide described hereinand that lacks a gene encoding a fatty acyl-CoA synthetase (FACS) and/ora gene encoding a fatty acyl-ACP thioesterase (TE). Without being boundto a particular theory, fatty alcohol production may be increased in amicroorganism lacking a gene encoding a FACS and/or a TE becausesilencing or inactivating the FACS and/or TE gene may inactivate acompeting biosynthetic pathway.

Accordingly, genetically modified E. coli host microorganisms silencedor inactivated in the fatty acyl-CoA synthetase fadD gene and/or theshort chain fatty acyl-CA synthetase fadK gene can be used asrecombinant hosts for production of fatty alcohols. The E. coli hostmicroorganism can be genetically modified to be silenced or inactivatedin one or more of the additional genes described above.

Transformation and Cell Culture

Polynucleotides of the invention, encoding FAR variants, may beintroduced into host cells for expression of the FAR variant. Thepolynucleotide may be introduced into the cell as a self-replicatingepisome (e.g., expression vector) or may be stably integrated into thehost cell DNA.

In some embodiments, a host cell is transformed with a polynucleotideencoding a FAR variant. In transformation, the polynucleotide that isintroduced into the host cell remains in the genome or on a plasmid orother stably maintained vector in the cell and is capable of beinginherited by the progeny thereof. Stable transformation is typicallyaccomplished by transforming the host cell with an expression vectorcomprising the polynucleotide of interest (e.g., the polynucleotideencoding the FAR variant) along with a selectable marker gene (e.g., agene that confers resistance to an antibiotic). Only those host cellswhich have integrated the polynucleotide sequences of the expressionvector into their genome will survive selection with the marker (e.g.,antibiotic). These stably transformed host cells can then be propagatedaccording to known methods in the art.

Methods, reagents and tools for transforming host cells describedherein, such as bacteria, yeast (including oleaginous yeast) andfilamentous fungi are known in the art. General methods, reagents andtools for transforming, e.g., bacteria can be found, for example, inSambrook et al (2001) Molecular Cloning: A Laboratory Manual, 3^(rd)ed., Cold Spring Harbor Laboratory Press, New York. Methods, reagentsand tools for transforming yeast are described in “Guide to YeastGenetics and Molecular Biology,” C. Guthrie and G. Fink, Eds., Methodsin Enzymology 350 (Academic Press, San Diego, 2002). Methods, reagentsand tools for transforming, culturing, and manipulating Y. lipolyticaare found in “Yarrowia lipolytica,” C. Madzak, J. M. Nicaud and C.Gaillardin in “Production of Recombinant Proteins. Novel Microbial andEucaryotic Expression Systems,” G. Gellissen, Ed. 2005, which isincorporated herein by reference for all purposes. In some embodiments,introduction of the DNA construct or vector of the present inventioninto a host cell can be effected by calcium phosphate transfection,DEAE-Dextran mediated transfection, PEG-mediated transformation,electroporation, or other common techniques (See Davis et al., 1986,Basic Methods in Molecular Biology, which is incorporated herein byreference).

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants, or amplifying the FAR polynucleotide. Culture conditions,such as temperature, pH and the like, are those previously used with thehost cell selected for expression, and will be apparent to those skilledin the art. As noted, many references are available for the culture andproduction of many cells, including cells of bacterial, plant, animal(especially mammalian) and archebacterial origin. See e.g., Sambrook,Ausubel, and Berger (all supra), as well as Freshney (1994) Culture ofAnimal Cells, a Manual of Basic Technique, third edition, Wiley-Liss,New York and the references cited therein; Doyle and Griffiths (1997)Mammalian Cell Culture: Essential Techniques John Wiley and Sons, NY;Humason (1979) Animal Tissue Techniques, fourth edition W.H. Freeman andCompany; and Ricciardelli, et al., (1989) In Vitro Cell Dev. Biol.25:1016-1024, all of which are incorporated herein by reference. Forplant cell culture and regeneration, Payne et al. (1992) Plant Cell andTissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg New York); Jones, ed. (1984) Plant Gene Transfer andExpression Protocols, Humana Press, Totowa, N.J. and Plant MolecularBiology (1993) R. R. D. Croy, Ed. Bios Scientific Publishers, Oxford,U.K. ISBN 0 12 198370 6, all of which are incorporated herein byreference. Cell culture media in general are set forth in Atlas andParks (eds.) The Handbook of Microbiological Media (1993) CRC Press,Boca Raton, Fla., which is incorporated herein by reference. Additionalinformation for cell culture is found in available commercial literaturesuch as the Life Science Research Cell Culture Catalogue (1998) fromSigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-LSRCCC”) and, for example,The Plant Culture Catalogue and supplement (1997) also fromSigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-PCCS”), all of which areincorporated herein by reference.

VII. Production and Recovery of Far Variants

In another aspect, the present invention provides a method of making apolypeptide having improved FAR enzymatic activity. In some embodiments,the method comprises: providing a host cell transformed with any one ofthe described FAR polynucleotides of the present invention; culturingthe transformed host cell in a culture medium under conditions in whichthe host cell expresses the encoded FAR polypeptide; and optionallyrecovering or isolating the expressed FAR polypeptide. The methodfurther provides optionally lysing the transformed host cells afterexpressing the encoded FAR polypeptide and optionally recovering orisolating the expressed FAR polypeptide from the cell lysate. Thepresent invention further provides a method of making an FARpolypeptide, said method comprising cultivating a host cell transformedwith a FAR polypeptide under conditions suitable for the production ofthe FAR polypeptide and recovering the FAR polypeptide.

The FAR polypeptide can be recovered from the host cell using proteinrecovery techniques that are well known in the art, including thosedescribed herein. Cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractmay be retained for further purification. Microbial cells employed inexpression of proteins can be disrupted by any convenient method,including freeze-thaw cycling, sonication, mechanical disruption, or useof cell lysing agents, or other methods, which are well known to thoseskilled in the art.

The resulting polypeptide may be recovered/isolated and optionallypurified by any of a number of methods known in the art. For example,the polypeptide may be isolated from the nutrient medium by conventionalprocedures including, but not limited to, centrifugation, filtration,extraction, spray-drying, evaporation, chromatography (e.g., ionexchange, affinity, hydrophobic interaction, chromatofocusing, and sizeexclusion), or precipitation. Protein refolding steps can be used, asdesired, in completing the configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed in thefinal purification steps. See, for example, Parry et al., 2001, Biochem.J. 353:117, and Hong et al., 2007, Appl. Microbiol. Biotechnol. 73:1331,both incorporated herein by reference. In addition to the referencesnoted supra, a variety of purification methods are well known in theart, including, for example, those set forth in Sandana (1997)Bioseparation of Proteins, Academic Press, Inc.; Bollag et al. (1996)Protein Methods, 2^(nd) Edition, Wiley-Liss, NY; Walker (1996) TheProtein Protocols Handbook Humana Press, NJ; Harris and Angal (1990)Protein Purification Applications: A Practical Approach, IRL Press atOxford, Oxford, England; Harris and Angal Protein Purification Methods:A Practical Approach, IRL Press at Oxford, Oxford, England; Scopes(1993) Protein Purification: Principles and Practice 3^(rd) Edition,Springer Verlag, NY; Janson and Ryden (1998) Protein Purification:Principles, High Resolution Methods and Applications, Second Edition,Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM, HumanaPress, NJ, all of which are incorporated herein by reference.

VIII. Methods of Producing Fatty Alcohols and Fatty Alcohol Compositions

The present disclosure also provides methods of producing fatty alcoholswith the improved FAR polypeptides described herein, as well as theresultant fatty alcohol compositions produced by said methods. Themethods can be carried out in cell-free systems with isolated improvedFAR polypeptides, or in cell-based systems with microorganismsengineered to express one or more improved FAR polypeptides, asdescribed above.

In embodiments in which fatty alcohols are produced in cell-freesystems, an isolated improved FAR polypeptide is provided with asubstrate (a fatty acyl-ACP and/or a fatty acyl-CoA complex) and NAD(P)Hunder suitable conditions of temperature, pH, and ionic strength andtime sufficient for the production of a fatty alcohol composition. Insome embodiments, the improved FAR polypeptide is provided with acomposition of a fatty acid, Coenzyme A and a fatty acyl-CoA synthaseunder suitable conditions of temperature, pH and ionic strength and timesufficient for production of a fatty alcohol composition.

In embodiments employing cell-based systems, a recombinant host cellcapable of expressing a gene that encodes an improved FAR polypeptide asdescribed herein above is cultured in an aqueous nutrient mediumcomprising an assimilable source of carbon under conditions suitable forproduction of a fatty alcohol composition. Any of the various hostmicroorganisms described herein can be used.

In some particular embodiments, a method of producing a fatty alcoholcomposition comprises culturing a recombinant microorganism in asuitable culture medium, wherein the recombinant microorganism comprisesa gene encoding an improved FAR polypeptide capable of producing atleast about 1.5 more fatty alcohol than a wild-type FAR comprising SEQID NO:2 when assayed under the same conditions. In some embodiments, amethod of producing a fatty alcohol composition comprises culturing arecombinant microorganism in a suitable culture medium, wherein therecombinant microorganism comprises a gene encoding a FAR variantcapable of producing an increased amount of C10-C18 fatty alcohols, anincreased amount of C12-C16 fatty alcohols, an increased amount ofC10-C14 fatty alcohols, an increased amount of C12-C14 fatty alcohols,or an increased amount of C16-018 fatty alcohols as compared to awild-type FAR comprising SEQ ID NO:2 when assayed under the sameconditions. In some embodiments, a method of producing a fatty alcoholcomposition comprises culturing a recombinant microorganism in asuitable culture medium, wherein the recombinant microorganism comprisesa gene encoding a FAR variant capable of producing a fatty alcoholprofile having one or more of an increased amount of C16:1 (cisΔ⁹-1-hexadecenol), a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol),an increased amount of C14:0 (1-tetradecanol), and a decreased amount ofC16:0 (1-hexadecanol) as compared to a wild-type FAR comprising SEQ IDNO:2.

In some embodiments, the method of producing a fatty alcohol compositioncomprises culturing a recombinant microorganism (for example, but notlimited to a strain of E. coli, Yarrowia, or Saccharomyces), in asuitable culture medium, wherein the recombinant microorganism comprisesa gene encoding an improved FAR polypeptide comprising a sequence thatis at least about 70% (or at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99%) identical to SEQ ID NO:2 and comprises one or more amino acidsubstitutions selected from A2D/F/G/H/I/P/N/Q/T/V/W, T3R, Q4R, Q5S, Q6P,Q7N, N8K/S, G9D/F, A10T, A12T/V, G14N/R/V/W, E17D, Q18I, K22E, V24I,L33V, I42L, G50S/V, L54P, R60H, H61R, P62S, A63R/Y, R65G/Q/Y, L69E/Q,E71K, A73K/V, S74K/P, S76K/N/R, V77A/I, H83R, E87G/V, T91I/R, L93V,H98P/R, T101L, G102C, V104I/M, S107C/L/W, G110D, L111S, T112A, P113D/L,R115A/H, R117D, A120V, G121H/S, Q122R, A125V, N128H, S132G, N134K/R/S,E137L, E138L/Q, D1400, A142V, K144Q, L148E, E151L, V1531, N160S, A162T,N174C, N177Q/R/T, Q180H/R, V185A/I, I186A/G/Y, P188A/I/M/S, T197P,D198Q, E202G, E204G, E205G/P, V207I/L, L209K/N, D212R, K213R, V217L,R2200, K224R, L226A/M, E227A/G/H/R/T, K229R, R236K, E237L,S244A/F/G/H/P, D245N, T246A, L257K, K260R/T, A261D, S263P, G264S, S266A,I269T, S283E/F/K/M/T/V, I287L, E288Q, V290I, A295T/V, A299T, E303G,V3051, S306F/H/N/W, V318I, I328T, L330V, S331V, L332S, A333T, S339G/V,G340P/S/V, Q341K, R342L, G350S, G351C, K359L, L364F/I, M365N, A366T/V,T370A/I, A374K/Y, D376P, Q377C/K/Y, Y380K/N/R, R381C, T384R, A389I/UM/V,D396G, V397I/L, V398Y, V399T, G400A/L, G401A/C/I/US/V, R403C/S, V404A,P405A/C/F/G/US/V/W, L406Y, A409V/W/Y, G410A/C/H/N/Q/R/S, A412C/F/M/V,M413L/R, A416L/V, Q418I/R/V/Y, E421I/L/N/P/R/S/V/Y, N427K,D429E/K/N/Q/R, T430H/I/R, R432C/Q, S433F/H/K/L/N/W, T436D/K/Q, I437V,F440L, A443T, P444S, Y446H, S452A/G/N, S458G/L/M/Q, R459H, L463E/T,D464G, V466E/Q/R, A472V, Q474R, L479Q, I484V, G487R/S/T/Y, N490S, E496A,K498A, L499A/H/I/N/P/R/S, Y500C/G/H/L/N/P/Q/R/S/W, S501 G/R,L502A/P/Q/R/S, A504G/R, A505K, D506G/S, T507A/G/P/R/S, R508D/G/H,K509D/E/G/H/N/R/S/Y, K510A/D/G/P/S/Y, A511G/I/K/P/Q/R/S/T, andA512K/S/T, wherein the amino acid positions are numbered with referenceto SEQ ID NO:2; and allowing expression of said gene, wherein saidexpression results in the production of a composition of fatty alcohols(e.g., a composition comprising an increased amount of an aggregate ofthe fatty alcohols C14:0 (1-tetradecanol), C16:1 (cis Δ⁹-1-hexadecenol),C16:0 (1-hexadecanol), C18:1 (cis Δ¹¹-1-octadecenol), and C18:0(1-octadecanol), or a composition comprising a fatty alcohol profilehaving one or more of an increased amount of C16:1 (cisΔ⁹-1-hexadecenol), a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol),an increased amount of C14:0 (1-tetradecanol), and a decreased amount ofC16:0 (1-hexadecanol) as compared to a wild-type FAR).

In other embodiments, the method of producing a fatty alcoholcomposition comprises culturing a recombinant microorganism (forexample, but not limited to a strain of E. coli, Yarrowia, orSaccharomyces), in a suitable culture medium, wherein the recombinantmicroorganism comprises a gene encoding an improved FAR polypeptidecomprising a sequence that is at least about 80% (at least 85%, at least90%, at least 95% or at least 97%) identical to SEQ ID NO: 2 andincluding a substitution at one or more positions 2, 134, 138, 188, 405and 511 when aligned to SEQ ID NO: 2; and allowing expression of saidgene, wherein said expression results in the production of a compositionof fatty alcohols. In some embodiments, the substitution at position 2is H, T, D, F, V, G, Q, P or I; the substitution at position 134 is R,K, or S; the substitution at position 138 is Q or L; substitution atposition 188 is S; the substitution at position 405 is V, S, F, G, C, L,S, A, or W; and the substitution at position 511 is T, P, G, S, K, Q, orR.

Fermentation

Fermentation of the recombinant host cell is carried out under suitableconditions and for a time sufficient for production of fatty alcohols.Conditions for the culture and production of cells, includingfilamentous fungi, bacterial and yeast cells, are readily available.Cell culture media in general are set forth in Atlas and Parks, Eds.,The Handbook of Microbiological Media (1993) CRC Press, Boca Raton,Fla., which is incorporated herein by reference. The individualcomponents of such media are available from commercial sources, e.g.,under the Difco™ and BBL™ trademarks. In one non-limiting example, theaqueous nutrient medium is a “rich medium” comprising complex sources ofnitrogen, salts, and carbon, such as YP medium, comprising 10 g/L ofpeptone and 10 g/L yeast extract of such a medium. In other non-limitingembodiments, the aqueous nutrient medium comprises a mixture of YeastNitrogen Base (Difco™) in combination supplemented with an appropriatemixture of amino acids, e.g., SC medium. In particular aspects of thisembodiment, the amino acid mixture lacks one or more amino acids,thereby imposing selective pressure for maintenance of an expressionvector within the recombinant host cell.

The recombinant microorganisms can be grown under batch or continuousfermentation conditions. Classical batch fermentation is a closedsystem, wherein the compositions of the medium is set at the beginningof the fermentation and is not subject to artificial alternations duringthe fermentation. A variation of the batch system is a fed-batchfermentation which also finds use in the present invention. In thisvariation, the substrate is added in increments as the fermentationprogresses. Fed-batch systems are useful when catabolite repression islikely to inhibit the metabolism of the cells and where it is desirableto have limited amounts of substrate in the medium. Batch and fed-batchfermentations are common and well known in the art. Continuousfermentation is an open system where a defined fermentation medium isadded continuously to a bioreactor and an equal amount of conditionedmedium is removed simultaneously for processing. Continuous fermentationgenerally maintains the cultures at a constant high density where cellsare primarily in log phase growth. Continuous fermentation systemsstrive to maintain steady state growth conditions. Methods formodulating nutrients and growth factors for continuous fermentationprocesses as well as techniques for maximizing the rate of productformation are well known in the art of industrial microbiology.

In some embodiments, fermentations are carried out a temperature withinthe range of from about 10° C. to about 60° C., from about 15° C. toabout 50° C., from about 20° C. to about 45° C., from about 20° C. toabout 40° C., from about 20° C. to about 35° C. and from about 25° C. toabout 45° C. In a particular aspect, the fermentation is carried out ata temperature of from about 28° C. and also from about 30° C. In otherembodiments, the fermentation is carried out for a period of time withinthe range of from about 8 hours to 240 hours, from about 8 hours toabout 168 hours, from about 8 hours to 144 hours, from about 16 hours toabout 120 hours, or from about 24 hours to about 72 hours. It will beunderstood that, in certain embodiments where thermostable host cellsare used, fermentations may be carried out at higher temperatures. Inother embodiments, the fermentation will be carried out at a pH in therange of 4-8, in the range of 4.5 to 7.5, in the range of 5 to 7, andalso in the range of 5.5 to 6.5. As used herein, the terms “culture” and“fermentation” are used interchangeably.

Carbon sources useful in the aqueous fermentation medium or broth of thedisclosed process in which the recombinant microorganisms are grown arethose assimilable by the recombinant host strain. Assimilable carbonsources are available in many forms and include renewable carbon sourcesand the cellulosic and starch feedstock substrates obtained there from.Such examples include, for example, depolymerized cellulosic material,monosaccharides, disaccharides, oligosaccharides, saturated andunsaturated fatty acids, succinate, acetate and mixtures thereof.Further carbon sources include, without limitation, glucose, galactose,sucrose, xylose, fructose, glycerol, arabinose, mannose, raffinose,lactose, maltose, and mixtures thereof. In some embodiments, the term“fermentable sugars” is used interchangeably with the term “assimilablecarbon source”. In one aspect, fermentation is carried out with amixture of glucose and galactose as the assimilable carbon source. Inanother aspect, fermentation is carried out with glucose alone toaccumulate biomass, after which the glucose is substantially removed andreplaced with an inducer, e.g., galactose for induction of expression ofone or more heterologous genes involved in fatty alcohol production. Instill another aspect, fermentation is carried out with an assimilablecarbon source that does not mediate glucose repression, e.g., raffinose,to accumulate biomass, after which the inducer, e.g., galactose, isadded to induce expression of one or more heterologous genes involved infatty alcohol production. In some preferred embodiments, the assimilablecarbon source is from cellulosic and starch feedstock derived from butnot limited to, wood, wood pulp, paper pulp, grain, corn stover, cornfiber, rice, paper and pulp processing waste, woody or herbaceousplants, fruit or vegetable pulp, distillers grain, grasses, rice hulls,wheat straw, cotton, hemp, flax, sisal, corn cobs, sugar cane bagasse,switch grass and mixtures thereof.

Production Levels

The methods described herein produce fatty alcohols in high yield. Cellsexpressing FAR variants described herein may yield fatty alcohols in therange of about 0.5 g to at least 35.0 g fatty alcohols per liter ofnutrient medium, depending upon the improved FAR polypeptide used.Exemplary culture conditions for E. coli are provided in the examples.Other E. coli culture conditions, as well as culture conditions forother host cells, are known or can be determined. In some embodiments,about 35 g/L to about 50 g/L (e.g., about 35 g/L, about 40 g/L, about 45g/L, or about 50 g/L), or sometimes about 50 g/L to about 100 g/L (e.g.,about 50 g/L, about 60 g/L, about 70 g/L, about 80 g/L, about 90 g/L, orabout 100 g/L) are produced. In particular embodiments, the amount offatty alcohols produced by the methods described herein is at leastabout 0.5 g/L, such as at least about 1 g/L, such as at least about 1.5g/L, such as at least about 2.0 g/L, such as at least about 2.5 g/L,such as at least about 3 g/L, such as at least about 3.5 g/L, such as atleast about 4 g/L, such as at least about 4.5 g/L, such as at leastabout 5 g/L, such as at least about 10 g/L of medium. In variousembodiments, the amount of fatty alcohols produced by the methodsdescribed herein is at least about 20 g/L, such as at least about 30g/L, such as at least about 40 g/L, such as at least about 50 g/L ofmedium. In some embodiments fermentation yields at least 0.1, at least0.15 or at least 0.18 g fatty alcohol/gram glucose. In some embodimentsfermentation yields at least 1 gram, at least 1.5 grams or at least 1.8grams fatty alcohol/gram dry cell weight.

In some embodiments, the methods described herein produce fatty alcoholcompositions of particular chain lengths in high yield. In someembodiments, the methods described herein produce fatty alcoholcompositions comprising at least about 90% C10-C18 fatty alcohols in anamount that is at least about 0.5 g/L, such as at least about 1 g/L, atleast about 1.5 g/L, at least about 2.0 g/L, at least about 2.5 g/L, atleast about 3 g/L, at least about 3.5 g/L, at least about 4 g/L, atleast about 4.5 g/L, at least about 5 g/L, at least about 10 g/L, atleast about 20 g/L, at least about 30 g/L, at least about 40 g/L, or atleast about 50 g/L fatty alcohols per liter of medium. In someembodiments, the methods described herein produce fatty alcoholcompositions comprising at least about 90% C12-C16 fatty alcohols in anamount that is at least about 0.5 g/L, such as at least about 1 g/L, atleast about 1.5 g/L, at least about 2.0 g/L, at least about 2.5 g/L, atleast about 3 g/L, at least about 3.5 g/L, at least about 4 g/L, atleast about 4.5 g/L, at least about 5 g/L, at least about 10 g/L, atleast about 20 g/L, at least about 30 g/L, at least about 40 g/L, or atleast about 50 g/L fatty alcohols per liter of medium. In someembodiments, the methods described herein produce fatty alcoholcompositions comprising at least about 90% C14-C16 fatty alcohols in anamount that is at least about 0.5 g/L, such as at least about 1 g/L, atleast about 1.5 g/L, at least about 2.0 g/L, at least about 2.5 g/L, atleast about 3 g/L, at least about 3.5 g/L, at least about 4 g/L, atleast about 4.5 g/L, at least about 5 g/L, at least about 10 g/L, atleast about 20 g/L, at least about 30 g/L, at least about 40 g/L, or atleast about 50 g/L fatty alcohols per liter of medium. In someembodiments, the methods described herein produce fatty alcoholcompositions comprising at least about 90% C16-C18 fatty alcohols in anamount that is at least about 0.5 g/L, such as at least about 1 g/L, atleast about 1.5 g/L, at least about 2.0 g/L, at least about 2.5 g/L, atleast about 3 g/L, at least about 3.5 g/L, at least about 4 g/L, atleast about 4.5 g/L, at least about 5 g/L, at least about 10 g/L, atleast about 20 g/L, at least about 30 g/L, at least about 40 g/L, or atleast about 50 g/L fatty alcohols per liter of medium. In someembodiments, the methods described herein produce an aggregate of thefatty alcohols C14:0 (1-tetradecanol), C16:1 (cis Δ⁹-1-hexadecenol),C16:0 (1-hexadecanol), C18:1 (cis Δ¹¹-1-octadecenol), and C18:0(1-octadecanol) in high yield. In some embodiments, the amount of suchan aggregate of fatty alcohols that is produced is at least about 0.5g/L, such as at least about 1 g/L, at least about 1.5 g/L, at leastabout 2.0 g/L, at least about 2.5 g/L, at least about 3 g/L, at leastabout 3.5 g/L, at least about 4 g/L, at least about 4.5 g/L, at leastabout 5 g/L, at least about 10 g/L, at least about 20 g/L, at leastabout 30 g/L, at least about 40 g/L, or at least about 50 g/L of medium.

In some embodiments, the amount of fatty alcohols produced by themethods described herein is in the range of about 100 mg/g to about 5g/g of dry cell weight. In other embodiments, the amount of fattyalcohols produced by the methods described herein is in the range ofabout 1 g/g to about 4 g/g of dry cell weight, such as in the range ofabout 2 g/g to about 3 g/g of dry cell weight by routine modification ofculturing conditions.

In certain embodiments, the amount of fatty alcohols produced by themethods described herein is in the range of about 10% to about 20% ofdry cell weight, such as in the range of about 20% to about 30% of drycell weight, such as in the range of about 30% to about 40% of dry cellweight, such as in the range of about 40% to about 50% of dry cellweight, such as in the in range of about 50% to about 60% of dry cellweight, such as in the range of about 60% to about 70% of dry cellweight, such as in the range of about 70% to about 80% of dry cellweight by routine modification of culturing conditions.

In some embodiments, host cells of the invention produce fatty alcoholcompositions having particular profiles as compared to the fatty alcoholcompositions produced by a wild-type FAR enzyme. In some embodiments,the methods of the present invention produce fatty alcohol compositionshaving an increased amount of C14:0 (1-tetradecanol) and/or an increasedamount of C16:1 (cis Δ⁹-1-hexadecenol) relative to the wild-type FARfrom which the FAR variant is derived. In some embodiments, the methodsof the present invention produce fatty alcohol compositions having adecreased amount of 018:0 (1-octadecanol) and/or a decreased amount ofC18:1 (cis Δ¹¹-1-octadecenol) relative to the wild-type FAR from whichthe FAR variant is derived. In some embodiments, the methods of thepresent invention produce fatty alcohol compositions having an increasedamount of C14:0 (1-tetradecanol) and a decreased amount of C18:1 (cisΔ¹¹-1-octadecenol) relative to the wild-type FAR from which the FARvariant is derived. For example, in some embodiments, the methods of thepresent invention produce fatty alcohol compositions having an increasedamount of C16:1 (cis Δ⁹-1-hexadecenol) relative to the wild-type FARfrom which the FAR variant is derived (e.g., increased by at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, or more relative to thewild-type FAR enzyme from which the FAR variant is derived). In someembodiments, the methods of the present invention produce fatty alcoholcompositions having a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol)relative to the wild-type FAR from which the FAR variant is derived(e.g., decreased by at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, or more relative to the wild-type FAR enzyme from which theFAR variant is derived). In some embodiments, the methods of the presentinvention produce fatty alcohol compositions having an increased amountof C14:0 (1-tetradecanol) relative to the wild-type FAR from which theFAR variant is derived (e.g., increased by at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, or more relative to the wild-type FARenzyme from which the FAR variant is derived). In some embodiments, themethods of the present invention produce fatty alcohol compositionshaving a decreased amount of C16:0 (1-hexadecanol) relative to thewild-type FAR from which the FAR variant is derived (e.g., decreased byat least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, or morerelative to the wild-type FAR enzyme from which the FAR variant isderived). In some embodiments, the methods of the present inventionproduce fatty alcohol compositions having a combination of two or moreof: an increased amount of C16:1 (cis Δ⁹-1-hexadecenol) (e.g., increasedby at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, or morerelative to the wild-type FAR enzyme from which the FAR variant isderived); a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol) (e.g.,decreased by at least about 5%, at least about 10%, at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,or more relative to the wild-type FAR enzyme from which the FAR variantis derived); an increased amount of C14:0 (1-tetradecanol) (e.g.,increased by at least about 5%, at least about 10%, at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,or more relative to the wild-type FAR enzyme from which the FAR variantis derived); and a decreased amount of C16:0 (1-hexadecanol) (e.g.,decreased by at least about 5%, at least about 10%, at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,or more relative to the wild-type FAR enzyme from which the FAR variantis derived).

Methods of Producing a Fatty Alcohol Composition

In another aspect, the present invention provides for methods ofproducing a fatty alcohol composition using a host cell comprising a FARvariant of the present invention. Fatty alcohol compositions can be madeby culturing a host cell comprising a FAR variant as described herein ina suitable culture medium under conditions (e.g., time, temperature,and/or pH conditions) suitable for the production of fatty alcohols, andproducing the fatty alcohol composition. In some embodiments, themethods further comprise isolating the fatty alcohol compositions fromthe culture medium. In some embodiments, the host cell comprising theFAR variant is a bacteria (e.g., E. coli), a yeast (e.g., Yarrowia orSaccharomyces), or a fungus. In some embodiments, the FAR variantcomprises at least about 70% identity to SEQ ID NO:2 and comprises oneor more amino acid substitutions as described herein (e.g., in Table 2,Table 4, Table 5, or Table 6). In some embodiments, the host cellcomprising the FAR variant is cultured under temperature conditions offrom about 10° C. to about 60° C. (e.g., from about 15° C. to about 50°C., from about 20° C. to about 45° C., from about 20° C. to about 40°C., from about 20° C. to about 35° C., or from about 25° C. to about 45°C.). In some embodiments, the host cell comprising the FAR variant iscultured under time conditions the fermentation of from about 8 hours to240 hours (e.g., from about 8 hours to about 168 hours, from about 8hours to 144 hours, from about 16 hours to about 120 hours, or fromabout 24 hours to about 72 hours). In some embodiments, the host cellcomprising the FAR variant is cultured under pH conditions of about pH4-8 (e.g., about pH 4.5 to 7.5, about pH 5 to 7, or about pH 5.5 to6.5).

While not meant to limit the invention in any manner, in someembodiments, the method of producing a fatty alcohol compositioncomprises

a) culturing a recombinant strain of E. coli, Yarrowia, orSaccharomyces, in a suitable culture medium, wherein the recombinantstrain comprises a gene encoding an improved FAR polypeptide,

b) allowing expression of said gene, and

c) producing the fatty alcohol composition, wherein i) the improved FARpolypeptide comprises an amino acid sequence that is at least about 70%identical to SEQ ID NO:2 and comprises one or more amino acidsubstitutions as described herein (e.g., one or more amino acidsubstitutions sets listed in Table 2, Table 4, or Table 5); ii) theculturing is carried at a temperature of about 20° C. to about 40° C.and from about 16 to 120 hours, iii) the culture medium comprises acarbon source comprising fermentable sugars obtained from a cellulosicfeedstock, and iv) at least about 5 g/L of recoverable fatty alcoholsare produced. In some embodiments, fermentable sugars in the culturemedium include glucose and/or sucrose.

In some embodiments, the method of producing a fatty alcohol compositioncomprises

a) culturing a recombinant strain of E. coli, Yarrowia, orSaccharomyces, in a suitable culture medium, wherein the recombinantstrain comprises a gene encoding an improved FAR polypeptide,

b) allowing expression of said gene, and

c) producing the fatty alcohol composition, wherein i) the improved FARpolypeptide comprises an amino acid sequence that is at least about 95%identical to SEQ ID NO:2 and comprises a substitution at one or morepositions 2, 134, 138, 188, 405 and 511 when aligned to SEQ ID NO:2, ii)the culturing is carried at a temperature of about 20° C. to about 40°C. and from about 16 to 120 hours, iii) the culture medium comprises acarbon source comprising fermentable sugars obtained from a cellulosicfeedstock, and iv) at least about 5 g/L of fatty alcohol are produced.In certain embodiments, the substitution at position 2 is H, T, D, F, V,G, Q, P or I; the substitution at position 134 is R, K, or 5; thesubstitution at position 138 is Q or L; substitution at position 188 is5; the substitution at position 405 is V, S, F, G, C, L, S, A, or W; andthe substitution at position 511 is T, P, G, 5, K, Q, or R, and theimproved FAR polypeptide will comprise one or more additionalsubstitutions of an amino acid residue relative to a positioncorresponding to SEQ ID NO:2.

Recovery of Fatty Alcohols

Fatty alcohols produced by the methods can be isolated to yield fattyalcohol compositions. In some embodiments, recombinant microorganismhosts secrete the fatty alcohols into the nutrient medium. Forcell-based methods carried out with recombinant microorganism hosts thatsecrete the fatty alcohols into the nutrient medium, the fatty alcoholscan be isolated by solvent extraction of the aqueous nutrient mediumwith a suitable water immiscible solvent. Phase separation followed bysolvent removal provides the fatty alcohol which may then be furtherpurified and fractionated using methods and equipment known in the art.In other aspects of the disclosure, the secreted fatty alcohols coalesceto form a water immiscible phase that can be directly separated from theaqueous nutrient medium either during the fermentation or after itscompletion.

In certain embodiments, fatty alcohols are isolated by separating thecells from the aqueous nutrient medium, for example by centrifugation,resuspension and extraction of the fatty alcohols from the recombinanthost cells using an organic solvent or solvent mixture. Suitableprotocols for recovering fatty alcohols from recombinant host cellsand/or culture medium are known to the skilled artisan.

Fatty alcohols produced with microorganism hosts that do not secrete thefatty alcohols into the nutrient medium can be recovered by first lysingthe cells to release the fatty alcohols and extracting the fattyalcohols from the lysate using conventional means. Reference is made toYeast Protocols Handbook, Clontech Laboratories, Inc., A Takara BioCompany, 1290 Terra Bella Ave., Mountain View, Calif. 94043, publishedJuly 2009, available online.

In some embodiments, the compositions produced by the methods describedherein comprise saturated fatty alcohols, unsaturated fatty alcohols, orboth saturated and unsaturated fatty alcohols. In various embodiments,the compositions produced by the methods described herein comprise bothsaturated and unsaturated fatty alcohols. In certain embodiments, theunsaturated fatty alcohols are monounsaturated fatty alcohols. In someembodiments, the fatty alcohol compositions comprise both saturated andunsaturated fatty alcohols, and the amount of unsaturated fatty alcoholsis less than about 30%, such as less than about 20%, such as less thanabout 10%, such as less than about 5%, such as less than about 1% of thefatty alcohols present in the composition. In other embodiments, thefatty alcohol compositions comprise both saturated and unsaturated fattyalcohols, and the amount of saturated fatty alcohols is less than about30%, such as less than about 20%, such as less than about 10%, such asless than about 5%, such as less than about 1% of the fatty alcoholspresent in the composition.

In some embodiments, the fatty alcohol compositions produced by themethods described herein comprise one or more alcohols selected from thegroup consisting of 1-octanol (C8:0), 1-decanol (C10:0), 1-dodecanol(C12:0), 1-tetradecanol (C14:0), 1-hexadecanol (C16:0), 1-octadecanol(C18:0), 1-icosanol (C20:0), 1-docosanol (C22:0), 1-tetracosanol(C24:0), cis Δ⁹-1-hexadecenol (C16:1), and cis Δ¹¹-1-octadecenol(C18:1). In some embodiments, the fatty alcohol compositions produced bythe methods described herein comprise 1-tetradecanol (C14:0),1-hexadecanol (C16:0), 1-octadecanol (C18:0), cis Δ⁹-1-hexadecenol(C16:1), and/or cis Δ¹¹-1-octadecenol (C18:1).

In some embodiments, C8 to C20 fatty alcohols comprise at least about80%, such as at least about 85%, such as at least about 90%, such as atleast about 92%, such as at least about 95%, such as at least about 97%,such as at least about 99% by weight of the total isolated fattyalcohols. In certain embodiments, 010 to C18 fatty alcohols compriseabout 80%, such as at least about 85%, such as at least about 90%, suchas at least about 92%, such as at least about 95%, such as at leastabout 97%, such as at least about 99% by weight of the total isolatedfatty alcohols. In certain embodiments, C14 to C18 fatty alcoholscomprise about 80%, such as at least about 85%, such as at least about90%, such as at least about 92%, such as at least about 95%, such as atleast about 97%, such as at least about 99% by weight of the totalisolated fatty alcohols. In some embodiments, C16 to C18 fatty alcoholscomprise at least about 80%, such as at least about 85%, such as atleast about 90%, such as at least about 91%, such as at least about 92%,such as at least about 93%, such as at least about 94%, such as at leastabout 95%, such as at least about 96%, such as at least about 97%, suchas at least about 98%, such as at least about 99% by weight of the totalisolated fatty alcohols. In some embodiments, the C16 to C18 fattyalcohols are saturated. In some embodiments, the 016 to C18 fattyalcohols are a mixture of saturated and unsaturated fatty alcohols.

IX. Fatty Alcohol Derivatives

Fatty alcohols produced using the methods disclosed herein can beconverted to a variety of commercially useful compounds, referred to asfatty alcohol derivatives. Without limitation, exemplary fatty alcoholderivatives include fatty acids, fatty aldehydes, fatty esters, waxesters, fatty acetates, ethoxylates, sulphates, phosphates, amines,alkanes, and alkenes. The fatty alcohol derivatives may be obtained fromfatty alcohols using either enzymatic or chemical methods. In someembodiments, total fatty alcohols produced in a fermentation arederivatized. Sometimes fatty alcohols produced in a fermentation arefractionated, and a fraction(s) is derivatized.

Alkane and/or Alkene Compositions

In some embodiments, the fatty alcohol compositions produced by themethods described herein can be reduced to yield alkanes and/or alkeneshaving the same carbon chain length as the fatty alcohol startingmaterials. Without being bound by any particular theory, the hydroxylgroup of an alcohol is a poor leaving group, and therefore, in principlea chemical moiety that binds to the oxygen atom of the hydroxyl group tomake it a better leaving group can be used to reduce the fatty alcoholsdescribed herein. In another embodiment, alkanes can be produced byhydrogenation of fatty alcohols or fatty acids.

Any method known in the art can be used to reduce the fatty alcoholsproduced according to the methods described herein. In some embodiments,reduction of fatty alcohols can be carried out chemically, for example,by a Barton deoxygenation (or Barton-McCombie deoxygenation), a two-stepreaction in which the alcohol is first converted to a methyl xanthate orthioimidazoyl carbamate, and the xanthate or thioimidazoyl carbamate isreduced with a tin hydride or trialkylsilane reagent under radicalconditions to produce the alkane and/or alkene. See J. J. Li, C.Limberakis, D. A. Pflum, Modern Organic Synthesis in the Laboratory(Oxford University Press, 2007) at pp. 81-83.

In some embodiments, reduction of fatty alcohols to the correspondingalkanes and/or alkenes can be accomplished using a microorganism thathas a biosynthetic pathway for reducing fatty alcohols. In certainembodiments, the microorganism is a bacterium. In specific embodiments,the bacterium is Vibrio furnissii strain M1. In some embodiments, thefatty alcohol compositions produced by the methods described herein arecontacted with the appropriate microorganism for reduction to alkanesand/or alkenes. In other embodiments, the fatty alcohol compositionsproduced by the methods described herein are contacted with membranefractions from the appropriate microorganism so that the reduction iscarried out in a cell free system. See, e.g., Park, 2005, J. Bacteriol.187(4):1426-1429.

In certain embodiments, alkanes and/or alkenes produced by the reductionof fatty alcohols described herein are isolated from the reactionmixture and unreduced fatty alcohol starting materials to produce acomposition that comprises substantially all alkanes and/or alkenes. Insome embodiments, the alkanes and/or alkenes produced by the reductionof fatty alcohols described herein and the unreacted fatty alcoholstarting materials are isolated from the reaction mixture to produce acomposition comprising alkanes and/or alkenes and fatty alcohols.

In certain embodiments, the resulting compositions comprise at leastabout 60% alkanes and/or alkenes, such as at least about 70% alkanesand/or alkenes, such as at least about 80% alkanes and/or alkenes, suchas at least about 85% alkanes and/or alkenes, such as at least about 90%alkanes and/or alkenes, such as at least about 92% alkanes and/oralkenes, such as at least about 95% alkanes and/or alkenes, such as atleast about 96% alkanes and/or alkenes, such as at least about 97%alkanes and/or alkenes, such as at least about 98% alkanes and/oralkenes, such as at least about 99% alkanes and/or alkenes by weight ofthe composition after reduction.

In other embodiments, the resulting compositions comprise at least about10% alkanes and/or alkenes, such as at least about 20% alkanes and/oralkenes, such as at least about 30% alkanes and/or alkenes, such as atleast about 40% alkanes and/or alkenes, such as at least about 50%alkanes and/or alkenes by weight of the composition after reduction.

In some typical embodiments, the compositions produced by the methodsdescribed herein comprise one or more alkanes selected from the groupconsisting of octane, decane, dodecane, tetradecane, hexadecane,octadecane, icosane, docosane, and tetracosane. In other typicalembodiments, the compositions produced by the methods described hereincomprise one or more alkenes selected from the group consisting ofoctane, decene, dodecene, tetradecene, hexadecene, octadecene, icosene,docosene, and tetracosene.

In typical embodiments, C8 to C20 alkanes and/or alkenes comprise atleast about 80%, such as at least about 85%, such as at least about 90%,such as at least about 92%, such as at least about 95%, such as at leastabout 97%, such as at least about 99% by weight of the total alkanesand/or alkenes in the composition. In certain embodiments, 010 to C18alkanes and/or alkenes comprise about 80%, such as at least about 85%,such as at least about 90%, such as at least about 92%, such as at leastabout 95%, such as at least about 97%, such as at least about 99% byweight of the total alkanes and/or alkenes in the composition.

In some embodiments, the C8 to C20 alkane and/or alkene compositionproduced by the methods of the disclosure, i.e. using an improved FARenzyme of the disclosure, preferentially comprises alkanes and/oralkenes of certain chain lengths. In one embodiment, 016:1 fatty alcoholis preferentially produced. In another embodiment, the compositioncomprises the following fatty alcohols in order of decreasing relativedistribution: C16:1, C16:0, C18:1, C14:0, and C18:0. In anotherembodiment, the composition comprises the following fatty alcohols inorder of decreasing relative distribution: C16:1, C16:0, C14:0, C18:1,and C18:0. In yet another embodiment, the composition comprises thefollowing fatty alcohols in order of decreasing relative distribution:C16:1, C14:0, C16:0, C18:1, and C18:0.

In certain embodiments, alkanes and/or alkenes having particular carbonchain lengths can be isolated from longer and/or shorter alkanes and/oralkenes, for example by HPLC. In certain embodiments, alkane and/oralkene compositions that are suitable, e.g., for use in jet fuels,comprise C10 to C14 alkanes and/or alkenes. In other embodiments, alkaneand/or alkene compositions that are suitable, e.g., for use in dieselfuels comprise alkanes and/or alkenes that have 16 or more carbons(e.g., C16 or longer-chain alkanes and/or alkenes).

Ester Compositions

In certain embodiments, the fatty alcohols are further processed with acarboxylic acid to form acid esters. Esterification reactions of fattyalcohols are well-known in the art. In certain embodiments, thetransesterification reaction is carried out in the presence of a strongcatalyst, e.g., a strong alkaline such as sodium hydroxide. In otherembodiments, the reaction is carried out enzymatically using an enzymethat catalyzes the conversion of fatty alcohols to acid esters, such aslipoprotein lipase. See, e.g., Tsujita et al., 1999, J. Biochem.126(6):1074-1079.

Sulfate Derivatives

In some embodiments, the fatty alcohols can be reacted with a sulfonicacid group to produce sulfate derivatives.

X. Exemplary Compositions Containing Fatty Alcohols and Fatty AlcoholDerivatives Produced According to the Methods of the Invention

In yet another aspect, the present invention relates to the use of FARvariants as described herein and microorganisms expressing the FARvariants as described herein for the production of various compositions,including but not limited to, detergent compositions (e.g., laundrydetergents in liquid and powder form, hard surface cleaners, dishwashingliquids, and the like); industrial compositions (e.g., lubricants,solvents; and industrial cleaners); personal care compositions (e.g.,soaps, cosmetics, shampoos, and gels); and fuel compositions (e.g.,biodiesels and petrodiesels). These compositions may comprise fattyalcohols and/or fatty alcohol derivatives.

Fuel Compositions

The fatty alcohol compositions described herein and compounds derivedthere from can be used as components of fuel compositions. In certainembodiments, the fatty alcohol compositions produced by the methodsdescribed above can be used directly in fuel compositions. Fuelcompositions containing fatty alcohols and derivatives produced by themethods of the present invention include any compositions used inpowering combustion engines, including but not limited to biodieselfuels and petrodiesel fuels (e.g., jet fuels and rocket fuels).

In some embodiments, the fuel composition is diesel fuel. Diesel fuel isany fuel used in diesel engines and includes both petrodiesel andbiodiesel. Petrodiesel is a specific fractional distillate of fossilfuel oil. It is comprised of about 75% saturated hydrocarbons and 25%aromatic hydrocarbons. Biodiesel is not derived from petroleum but fromvegetable oil or animal fats and contains long chain alkyl esters.Biodiesel is made by the transesterification of lipids (e.g., spentvegetable oil from fryers or seed oils) with an alcohol and burnscleaner than petrodiesel. Biodiesel can be used alone or mixed withpetrodiesel in any amount for use in modern engines.

In some embodiments, the fuel composition is kerosene. Kerosene is acombustible hydrocarbon that is also a specific fractional distillate offossil fuel and contains hydrocarbons having 6 to 16 carbon atoms.Kerosene has a heat of combustion comparable to that of petrodiesel andis widely used in jet fuel to power jet engines and for heating incertain countries. Kerosene-based fuels can also be burned with liquidoxygen and used as rocket fuel (e.g., RP-1).

In various embodiments, the fatty alcohols can be reacted with acarboxylic acid to produce acid esters. In particular embodiments, theacid esters are used as components of biodiesel fuel compositions. Inother embodiments, the fatty alcohols are reacted with a reducing agentto produce alkanes and/or alkenes. In some embodiments, alkanes and/oralkenes (e.g., C10 to C14) derived from the fatty alcohol compositionsare used as components of jet fuel compositions. In other embodiments,alkanes and/or alkenes derived from fatty alcohol compositions are usedas components of rocket fuel. In still other embodiments, alkanes and/oralkenes (e.g., C16 to C24) derived from the fatty alcohol compositionsare used as components in petrodiesel-like fuel compositions.

In some embodiments, the fuel compositions comprise an alkane and/oralkene derived from the fatty alcohol compositions described herein. Incertain embodiments, the alkanes and/or alkenes have from 6 to 16carbons and the fuel composition is a kerosene-like fuel composition. Invarious embodiments, the kerosene-like fuel compositions are included injet fuel compositions. In particular embodiments, the kerosene-like fuelcompositions are included in various grades of jet fuel, including butnot limited to, grades Avtur, Jet A, Jet A-1, Jet B, JP-4, JP-5, JP-7and JP-8. In other embodiments, the kerosene-like fuel compositions areincluded in fuel compositions for heating. In still other embodiments,the kerosene-like fuel compositions derived from the fatty alcoholcompositions described above are burned with liquid oxygen to providerocket fuel. In particular embodiments, the kerosene-like fuelcompositions are used in RP-1 rocket fuel.

In some embodiments, the alkanes and/or alkenes derived from the fattyalcohol compositions described herein are used in fuel compositions thatare similar to petrodeisel fuel compositions, e.g., that containsaturated and aromatic hydrocarbons. In certain embodiments, the fuelcompositions comprise only alkanes and/or alkenes derived from the fattyalcohol compositions described herein. In other embodiments, the fuelcompositions comprise alkanes and/or alkenes derived from the fattyalcohol compositions described herein mixed with other components, suchas petrodiesel fuel. In some embodiments, the acid esters derived fromthe fatty alcohol compositions described herein are used as biodieselfuel without being mixed with other components. In other embodiments,the fatty acid esters derived from the fatty alcohol compositionsdescribed herein are mixed with other components, such as petrodieselfuel.

In certain embodiments, fatty alcohols, or acid esters or alkanes and/oralkenes derived there from, are combined with other fuels or fueladditives to produce compositions having desired properties for theirintended use. Exemplary fuels and fuel additives for particularapplications are well-known in the art. Exemplary fuels which can becombined with the compositions described herein include, but are notlimited to, traditional fuels such as ethanol and petroleum-based fuels.Exemplary fuel additives which can be combined with the compositionsdescribed herein include, but are not limited to, cloud point loweringadditives, surfactants, antioxidants, metal deactivators, corrosioninhibitors, anti-icing additives, anti-wear additives, deposit-modifyingadditives and octane enhancers.

Detergent Compositions

In certain embodiments, the fatty alcohol compositions described hereinand compounds derived there from can be used as components of detergentcompositions. Detergent compositions containing fatty alcohols producedby the methods of the present invention include compositions used incleaning applications, including, but not limited to, laundrydetergents, hand-washing agents, dishwashing detergents, rinse-aiddetergents, household detergents, and household cleaners, in liquid,gel, granular, powder, or tablet form. In some embodiments, the fattyalcohol compositions produced by the methods described above can be useddirectly in detergent compositions. In some embodiments, the fattyalcohols can be reacted with a sulfonic acid group to produce sulfatederivatives that can be used as components of detergent compositions.Detergent compositions that can be generated using the fatty alcoholcompositions produced by the methods of the present invention include,but are not limited to, hair shampoos and conditioners, carpet shampoos,light-duty household cleaners, light-duty household detergents,heavy-duty household cleaners, and heavy-duty household detergents.Detergent compositions generally include, in addition to fatty alcohols,one or more or of builders (e.g., sodium carbonate, complexation agents,soap, and zeolites), enzymes (e.g., a protease, a lipase and anamylases); carboxymethyl cellulose, optical brighteners, fabricsofteners, colourants and perfumes (e.g., cyclohexyl salicylate).

In some embodiments, sulfate derivatives (e.g., C12-15) derived from thefatty alcohol compositions are used in products such as hair shampoos,carpet shampoos, light-duty household cleaners, and light-duty householddetergents. In some embodiments, fatty alcohol compositions (e.g.,C16-C18) produced by the methods described herein are used in productssuch as hair shampoos and conditioners. In some embodiments, sulfatederivatives (e.g., C16-18) derived from the fatty alcohol compositionsare used in products such as heavy-duty household cleaners andheavy-duty household detergents.

Personal Care Compositions

In certain embodiments, the fatty alcohol compositions described hereinand compounds derived there from can be used as components of personalcare compositions. In some embodiments, the fatty alcohol compositionsproduced by the methods described above can be used directly in personalcare compositions. Personal care compositions containing fatty alcoholsproduced by the methods of the present invention include compositionsused for application to the body (e.g., for application to the skin,hair, nails, or oral cavity) for the purposes of grooming, cleaning,beautifying, or caring for the body, including but not limited tolotions, balms, creams, gels, serums, cleansers, toners, masks,sunscreens, soaps, shampoos, conditioners, body washes, styling aids,and cosmetic compositions (e.g., makeup in liquid, cream, solid,anhydrous, or pencil form). In some embodiments, the fatty alcohols canbe reacted with a sulfonic acid group to produce sulfate derivativesthat can be used as components of said compositions.

In some embodiments, fatty alcohol compositions (e.g., C12) produced bythe methods described herein are used in products such as lubricatingoils, pharmaceuticals, and as an emollient in cosmetics. In someembodiments, fatty alcohol compositions (e.g., C14) produced by themethods described herein are used in products such as cosmetics (e.g.,cold creams) for its emollient properties. In some embodiments, fattyalcohol compositions (e.g., C16) produced by the methods describedherein are used in products such as cosmetics (e.g., skin creams andlotions) as an emollient, emulsifier, or thickening agent. In someembodiments, fatty alcohol compositions (e.g., C18) produced by themethods described herein are used in products such as lubricants,resins, perfumes, and cosmetics, e.g., as an emollient, emulsifier, orthickening agent. In some embodiments, sulfate derivatives (e.g.,C12-14) derived from the fatty alcohol compositions produced by themethods described herein are used in products such as toothpastes.

Other Compositions

In some instances, fatty alcohols (especially cetyl alcohol, stearylalcohol and myristyl alcohol) may be used as food additives (e.g.,adjuvants and production aids).

XI. EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1 Wild-type M. algicola DG893 FAR Gene Acquisition and VectorConstruction

Gene acquisition of wild-type M. algicola DG893 FAR (“FAR Maa”) isdescribed in the published application WO/2011/008535. The genomicsequence of M. algicola DG893 can also be found at GenBank Accession No.NZ_ABCP01000001.1. The amino acid sequence of the encoded FARpolypeptide is designated SEQ ID NO:2. The polynucleotide sequences oftwo different codon-optimized genes encoding the FAR polypeptide of SEQID NO: 2 are designated SEQ ID NO:1 and SEQ ID NO:3. The M. algicolaDG893 FAR gene and genes encoding variants of the M. algicola DG893 FARwere cloned into the vector pCK110900 (depicted as FIG. 3 in U.S. PatentAppln. Pub. 2006/0195947) under the control of a lac promoter, asdescribed in WO 2011/008535. The resulting plasmids were introduced intoE. coli BW25113, BW25113 ΔfadE, or BW25113 ΔtorR (Baba et al., MolecularSystems Biology, 2006 doi:10,1038/msb4100050 Article No. 2006.0008),W3110-ΔfhuA and MG1655-7740 by routine transformation methods.

Example 2 Evaluation of M. algicola FAR Variants with Improved ActivityUsing Microtiter Plate Assays

FAR Maa Variant Nos. 1-172 were grown in 96-well shallow platescontaining 180 μL Luria Bertani (LB) or M9YE medium supplemented with 1%glucose and 30 μg/mL chloramphenicol (CAM), for approximately 16-18hours (overnight) in a shaker-incubator at 30° C., 200 rpm. A 5%inoculum was used in 96-deep-well plates to initiate fresh 380 μLculture containing 2×YT broth medium supplemented with 30 μg/mL CAM and0.4% glucose. The culture was incubated for 2 hours at 30° C., 250 rpmto an OD₆₀₀ of 0.6-0.8, at which point expression of the heterologousFAR gene was induced with isopropyl-β-D-thiogalactoside (IPTG) (1 mMfinal concentration). Incubation was continued for about 24 hours underthe same conditions. FAR Maa Variant Nos. 173-423 were grown under thesame conditions except M9YE medium was used to inoculate the cells andan additional amount of glucose (0.5% w/v final conc.) was added to theculture at 3 and 6 hrs after induction by IPTG. FAR Maa Variant Nos.424-527 were grown under the above conditions except M9YE mediumcontaining 5% glucose was used to inoculate the cells and induction byIPTG, the culture was incubated at 30° or 37° C. for 48 hours. FAR MaaVariant Nos. 528-629 were grown under the above conditions except M9YEmedium containing 10% glucose was used to inoculate the cells and afterinduction by IPTG, the culture was incubated at 40° for 24 hours.

Cell cultures were extracted with 1 mL of isopropanol:methyl t-butylether (MTBE) (4:6 ratio) for 2 hours. The extracts were centrifuged andthe upper organic phase was transferred into polypropylene 96-wellplates and analyzed by the following GC-FID method using DB-5MS column(length 30 m, I.D. 0.32 mm, film 0.25 um): start temp. 150° C., increasethe temperature at a rate of 25° C./min to 246° C. and hold for 1.81min. Total run time, 5.65 min. Under the above GC conditions theapproximate retention times (min) of produced fatty alcohols and acidswere as follows: 3.19, C14:0-OH; 3.48, C14:0-OOH; 3.91, C16:1-OH; 3.98,C16:0-OH; 4.15, C16:0-OOMe (internal standard); 4.21, C16:1-OOH; 4.28,C16:0-OOH; 4.83, C18:1-OH; 4.92, C18:0-OH; 5.31, C18:0-OOH and 5.51,C18:1-OOH. Identification of individual fatty alcohol was done bycomparison to commercial standards (Sigma Chemical Company, 6050 SpruceSt. Louis, Mo. 63103).

Table 2 provides the relative fatty alcohol production for illustrativevariants relative to wild-type M. algicola DG893 FAR. Codon-optimizedSEQ ID NO:3 was mutated and used to express FAR Maa Variant Nos. 1-172,and codon-optimized SEQ ID NO:1 was mutated and used to express FAR MaaVariants Nos. 173-629. Relative fatty alcohol production is presented asfold improvement over wild-type FAR Maa at 30° C. (for Variant Nos.1-423); over FAR Maa Variant No. 391 at 37° C. (for Variant Nos.424-527); or over FAR Maa Variant No. 438 at 40° C. (for Variant Nos.528-629). In Table 2, the amino acid substitutions listed for eachvariant correspond to residue positions of SEQ ID NO:2 (e.g., “G410S”means that the residue at position 410 in SEQ ID NO:2 (glycine) issubstituted with serine), and the amino acid positions were determinedby optimal alignment with SEQ ID NO:2. For Variant Nos. 424-527, theactivity of these variants was also tested at 30° C., and the activityof these variants at 30° C. was similar to that obtained from VariantNo. 391 at 30° C.

TABLE 2 Variant FAR polypeptides and total relative production of fattyalcohols Relative fold improvement in fatty Variant Amino acidsubstitutions alcohol No. relative to SEQ ID NO: 2 production† 1G410S; + 2 S283V; + 3 D198Q; + 4 S283K; + 5 S339V; + 6 S458G; + 7L463T; + 8 G410R; ++ 9 E288Q; + 10 G410Q; + 11 E138Q; + 12 S283M; + 13R236K; + 14 L33V; + 15 M413L; + 16 I287L; + 17 A416V; + 18 R117D; + 19E138L; K144Q; + 20 K359L; A511T; +++ 21 N134R; + 22 L209N; A412V; + 23S244G; + 24 H83R; + 25 A412V; + 26 E237L; ++ 27 L209N; + 28 T101L; + 29E151L; + 30 L463E; + 31 I437V; A511T; + 32 E204G; + 33 R65Q; + 34G340V; + 35 S458M; + 36 A443T; + 37 L257K; + 38 A374K; + 39 I42L; + 40S339G; + 41 N134K; ++ 42 V397L; + 43 S458Q; +++ 44 G487Y; + 45 G340S;P405S; + 46 A412F; + 47 A412C; + 48 R115A; + 49 E421R; +++ 50 P405S; +51 D140C; +++ 52 P405F; + 53 G14W; + 54 D429Q; + 55 S433K; ++ 56 N8K; +57 P405G; + 58 S501G; + 59 K229R; + 60 Q377C; + 61 Y380R; + 62 K509S; +63 Q418I; + 64 D506S; + 65 K510G; +++ 66 Y500C; + 67 E496A; + 68 A2H;+++ 69 P405C; + 70 A505K; + 71 G9F; + 72 S306W; + 73 E303G; T430I; + 74A504G; + 75 Q418V; + 76 Y500P; + 77 D506G; + 78 E421Y; ++ 79 S433H; + 80K510P; ++ 81 L502Q; + 82 A511P; ++ 83 A511G; +++ 84 L502A; + 85 S331V; +86 Q418R; + 87 A2W; +++ 88 E421I; ++ 89 A2D; +++ 90 K224R; + 91 Q418Y; +92 G401V; + 93 L502R; ++ 94 K510Y; + 95 L502S; + 96 Y500L; + 97 A504R;++ 98 E137L; + 99 S433W; + 100 T507G; + 101 Q180R; T246A; + 102 G401L; +103 G121S; S433L; + 104 K510S; + 105 T507A; + 106 S76K; + 107 K509H; ++108 K510D; + 109 A511R; +++ 110 G401S; + 111 S107L; ++ 112 R508G; + 113Q7N; + 114 Y500W; + 115 Q377Y; + 116 G400L; + 117 L499P; + 118 S74K; +119 T507R; + 120 E303G; + 121 A511T; ++++ 122 P113L; E421P; ++ 123 A2T;L332S; +++ 124 T3R; + 125 L499A; + 126 A2F; +++ 127 A511S; +++ 128A511K; +++ 129 P113D; + 130 S501R; + 131 L364F; G400A; + 132 A374Y; +133 Y500G; + 134 A511Q; ++++ 135 E421S; ++ 136 G14N; + 137 K509E; + 138G121H; + 139 D429K; ++ 140 A511I; + 141 A2V; ++ 142 Y500N; + 143 S433F;++ 144 L499H; + 145 T436K; + 146 A2V; S107C; ++ 147 A2G; L148E; +++ 148E205P; A512T; ++++ 149 A416L; + 150 T384R; + 151 A2Q; +++ 152 K498A;L502R; + 153 Y500Q; + 154 Y500S; + 155 P405L; + 156 T507S; + 157L499S; + 158 S306F; + 159 L226A; + 160 E17D; + 161 N8S; + 162 K509G; ++163 A73K; + 164 K510A; + 165 S433N; + 166 Q5S; P405S; + 167 L502P; + 168A2P; + 169 E421L; +++ 170 L499R; ++ 171 E421V; ++ 172 E421N; + 173 G50V;A511T; + 174 R381C; A511T; + 175 A142V; A511T; + 176 P188S; A511T; + 177S74P; + 178 A472V; A511T; ++ 179 V185A; A511T; ++ 180 P444S; A511T; +181 R459H; D464G; L499P; A511T; + 182 V77A; A511T; + 183 K260T; A511T; +184 H98R; A511T; + 185 K22E; A511T; + 186 V24I; R403C; A511T; + 187A125V; A511T; ++ 188 A299T; A511T; + 189 R220C; A511T; + 190 V185A;A333T; A511T; ++ 191 S458L; A511T; + 192 L111S; A511T; + 193 E71K;S458L; A511T; + 194 L93V; + 195 Y446H; A511T; + 196 D245N; A511T; ++ 197I328T; A511T; + 198 S263P; G410S; A511T; ++ 199 N490S; A511T; ++ 200E138Q; P188S; E227G; E237L; + 201 N134R; E138Q; P188S; ++ 202 E138Q;P188S; + 203 A120V; N134K; S458Q; A511T; + 204 Q4R; A10T; E138Q; ++ 205T91I; N134R; P188S; K260R; A511T; + 206 N134K; E138Q; P188S; S458Q; ++207 S458Q; I484V; + 208 N134K; E227G; + 209 N134S; E138Q; P188S; + 210T112A; N134K; P188S; + 211 N134S; E138Q; P188S; A511T; ++ 212 N134K;P188S; + 213 A12V; S458Q; A511T; ++ 214 N134R; G410Q; A412C; S458Q; +215 G102C; N134R; E138Q; P188S; + 216 N134S; E138Q; E205G; A511T; + 217N134S; E138Q; P188S; F440L; A511T; + 218 N134R; E202G; K213R; + 219N134R; P188S; K213R; K260R; I437V; S458Q; + 220 N134S; P188S; G410Q;A412V; S458Q; A511T; + 221 N134R; P188S; + 222 L54P; A366T; G410S;S458Q; A511T; + 223 N134S; E227G; + 224 G50S; N134K; E138Q; S458Q; ++225 S458Q; A511T; ++ 226 N134S; E138Q; P188S; E227G; + 227 E138Q; I269T;A511T; ++ 228 D396G; A511T; ++ 229 R115H; N134R; P188S; + 230 N134R;E138Q; N160S; P188S; E303G; ++ 231 R60H; T112A; N134R; E227G; V290I;G410Q; ++ I437V; S458Q; 232 E138Q; G350S; A511T; ++ 233 P188S; E227G; +234 E138Q; P188S; S306N; + 235 N134K; P188S; S458Q; ++ 236 N134K; S458L;++ 237 S132G; S458Q; A511T; + 238 A2G; N134S; E138Q; P188S; A511T; +++239 A2I; T112A; N134K; E138Q; P188S; A511T; + 240 N134S; E138Q; P188S;E227G; A511T; +++ 241 T112A; N134K; E138Q; P188S; A511T; ++ 242 A2G;T112A; N134R; E138Q; P188S; K510P; + A511R; 243 A2H; T112A; N134S;E138Q; P188S; S458Q; + A511T; 244 A2D; T112A; N134R; E138Q; P188S;E421V; ++ K509H; A511Q; A512T; 245 N134S; E138Q; P188S; V207I; K510P;A511G; +++ 246 N134S; E138Q; P188S; S458Q; K510P; A511K; +++ 247 Q122R;N134S; E138Q; P188S; K510P; A511R; +++ 248 N134S; E138Q; P188S; S458Q;K510P; A511R; +++ 249 N134S; E138Q; P188S; K510P; A511R; A512T; +++ 250N134S; E138Q; P188S; E227G; S458Q; K510P; +++ A511K; 251 A2G; T112A;N134K; E138Q; P188S; S458Q; ++ K509H; K510P; A511Q; 252 N134S; E138Q;P188S; E421S; S458Q; K509H; +++ K510P; A511R; 253 T112A; N134R; E138Q;P188S; E421R; S458Q; ++ K510P; A511S; 254 N134S; E138Q; P188S; K510P;A511G; A512T; +++ 255 A2H; N134S; E138Q; P188S; K510P; A511S; ++ A512T;256 A2W; T112A; N134R; E138Q; P188S; K509H; ++ K510P; A511G; 257 A2N;N134S; E138Q; P188S; E421V; A511T; + 258 N134S; E138Q; P188S; E421R;S458Q; K510P; +++ A511S; A512T; 259 N134S; E138Q; P188S; E421L; K509H;K510P; ++ A511K; 260 A2P; N134S; E138Q; P188S; K510P; A511R; +++ A512T;261 N134K; E138Q; P188S; A511T; ++ 262 T112A; N134S; E138Q; P188S;A511T; ++ 263 N134S; E138Q; P188S; S458Q; A511T; A512T; +++ 264 A2G;A73V; T112A; N134S; E138Q; P188S; +++ E227G; A511T; 265 E138Q; P188S;A511T; A512T; +++ 266 N134R; E138Q; P188S; A511T; ++ 267 N134S; E138Q;P188S; E421L; S458Q; A511T; ++ 268 N134S; E138Q; P188S; S458Q; A511T; ++269 N134S; E138Q; P188S; E421R; S458Q; A511T; ++ 270 T112A; E138Q;P188S; A511T; ++ 271 N134S; E138Q; P188S; E227G; A511T; A512T; +++ 272T112A; N134S; E138Q; P188S; E205P; E421R; + A511T; 273 A2G; T112A;E138Q; P188S; A511T; +++ 274 N134S; E138Q; P188S; A511T; A512T; ++ 275N134S; E138Q; P188S; D429N; L499S; K509G; ++ A511T; 276 N134S; E138Q;P188S; A412V; D429K; L499H; +++ K509E; A511T; 277 N134S; E138Q; P188S;D429N; K509N; A511T; +++ 278 N134S; E138Q; P188S; A412V; D429K; A511T; +279 N134S; E138Q; P188S; A412V; D429K; L499N; ++ A511T; 280 N134S;E138Q; P188S; L499R; K509R; A511T; ++ 281 N134S; E138Q; P188S; D429E;L499N; K509G; + A511T; 282 N134S; E138Q; Q180R; P188S; S306W; L499R; +++K509G; A511T; 283 G121S; N134S; E138Q; P188S; S306W; A374Y; +++ L499R;K509S; A511T; 284 N134S; E138Q; P188S; D429K; L499N; A504G; + K509G;A511T; 285 V77I; N134S; E138Q; P188S; S306W; A511T; +++ 286 N134S;E138Q; P188S; D429N; L499R; A504G; ++ K509G; A511T; 287 N134S; E138Q;P188S; D429E; L499N; A511T; +++ 288 N134S; E138Q; P188S; A412V; D429E;L499I; +++ K509D; A511T; 289 N134S; E138Q; P188S; S306W; A412V; D429K;+++ L499S; A511T; 290 N134S; E138Q; P188S; D429N; L499R; K509S; + A511T;291 N134S; E138Q; P188S; A412V; L499I; K509G; +++ A511T; 292 N134S;E138Q; P188S; D429E; L499H; K509G; +++ A511T; 293 N134S; E138Q; P188S;A504R; K509N; A511T; +++ 294 N134S; E138Q; P188S; A412V; D429E; L499R;++ K5090; A511T; 295 N134S; E138Q; P188S; D429N; L499I; K509G; + A511T;296 N134S; E138Q; P188S; S306W; A374Y; L499R; ++ A504R; A511T; 297N134S; E138Q; P188S; D429E; K509D; A511T; +++ 298 N134S; E138Q; Q180R;P188S; S306W; L499H; +++ K509S; A511T; 299 N134S; E138Q; P188S; V397I;A511T; +++ 300 G9F; N134S; E138Q; P188S; L502Q; +++ T507A; A511T; 301N134S; E138Q; P188S; G401V; L502S; A511T; ++++ 302 N134S; E138Q; P188S;G401S; A511T; +++ 303 G9F; P113L; N134S; E138Q; P188S; G401L; ++++L502S; T507A; A511T; 304 N134S; E138Q; P188S; G401L; A511T; ++++ 305G9F; N134S; E138Q; P188S; E288Q; G410R; + E421S; A511T; 306 P113L;N134S; E138Q; P188S; K224R; A366V; ++ L502Q; T507R; A511T; 307 N134S;E138Q; P188S; G401V; G487Y; L502S; +++ T507A; A511T; 308 N134S; E138Q;P188S; S244P; E288Q; G401S; ++++ A511T; 309 N134S; E138Q; P188S; G401L;G410R; G487Y; +++ L502S; A511T; 310 G9F; E87V; N134S; E138Q; P188S;K224R; +++ E288Q; G401V; G487Y; L502R; A511T; 311 N134S; E138Q; P188S;E421N; T507A; A511T; + 312 G9F; N134S; E138Q; P188S; K224R; E288Q; +++G401A; L502S; T507P; A511T; 313 P113L; N134S; E138Q; P188S; G487Y;L502S; +++ T507A; A511T; 314 G9F; P113L; N134S; E138Q; P188S; E288Q; +++G401L; A511T; 315 P113L; N134S; E138Q; P188S; L502Q; T507R; +++ A511T;316 N134S; E138Q; P188S; V404I; G410R; R508H; +++ A511T; 317 G9F; P113L;N134S; E138Q; P188S; G487Y; +++ L502A; A511T; 318 N134S; E138Q; P188S;G401S; G410R; A511T; +++ 319 N134S; E138Q; P188S; G401V; A511T; ++++ 320G9F; P113L; N134S; E138Q; P188S; G401A; +++ G410R; A511T; 321 G9F;P113L; N134S; E138Q; P188S; G401V; +++ T507A; A511T; 322 P113L; N134S;E138Q; P188S; K224R; G487Y; +++ L502R; A511T; 323 P113L; N134S; E138Q;P188S; E421S; G487Y; +++ L502A; A511T; 324 N134S; E138Q; P188S; A409V;A511T; +++ 325 S76K; N134S; E138Q; P188S; Y380R; A416L; +++ Y500N;S501R; R508G; A511T; 326 S76N; N134S; E138Q; P188S; Y380R; Y500N; ++S501R; A511T; 327 N134S; E138Q; P188S; Y500Q; S501R; A511T; ++ 328N134S; E138Q; P188S; L209N; Y380R; A409V; +++ A416L; T430I; Y500N;S501R; A511T; 329 S76R; N134S; E138Q; P188S; E303G; T430I; +++ Y500Q;S501G; R508G; A511T; 330 N134S; E138Q; P188S; L209N; E303G; R508G; +++A511T; 331 S76K; N134S; E138Q; P188S; E303G; Y380R; ++ T430I; Y500N;A511T; 332 S76N; N134S; E138Q; P188S; Y380R; A416L; +++ Y500Q; R508G;A511T; 333 N134S; E138Q; P188S; A416L; T430I; Y500G; +++ S501G; A511T;334 N134S; E138Q; P188S; R508G; A511T; +++ 335 N134S; E138Q; P188S;E303G; A511T; +++ 336 S76R; N134S; E138Q; P188S; A416L; T430I; +++Y500N; S501R; R508G; A511T; 337 N134S; E138Q; P188S;Y380R; A416L; T430I;+++ Y5000; S501G; R508G; A511T; 338 N134S; E138Q; V185I; P188S; A416L;Y500Q; +++ R508G; A511T; 339 N134S; E138Q; L148E; P188S; E303G; Y380R;+++ T430I; Y500Q; A511T; 340 N134S; E138Q; L148E; P188S; Y500Q ;S501R;++ A511T; 341 N134S; E138Q; P188S; A416L; A511T; +++ 342 N134S; E138Q;P188S; A416L; R508G; A511T; +++ 343 N134S; E138Q; P188S; Y380R; A511T ++344 S76K; N134S; E138Q; P188S; E303G; A416L; +++ T430I; Y500Q; A511T;345 N134S; E138Q; P188S; Y380R; Y500Q; R508G; +++ A511T; 346 N134S;E138Q; L148E; P188S; E303G; Y380R; +++ Y500N; A511T; 347 S76K; N134S;E138Q; P188S; A416L; A511T; +++ 348 N134S; E138Q; P188S; Y380R; T430I;Y500Q; +++ A511T; 349 N134S; E138Q; P188S; Y380R; A416V; R508G; +++A511T; 350 N134S; E138Q; P188S; Y500Q; S501G; R508G; +++ A511T; 351N134S; E138Q; P188S; L332S; P405F; A511T; +++ 352 N134S; E138Q; P188S;Q418R; S433N; K510D; +++ A511T; 353 N134S; E138Q; P188S; P405L; A511T;+++ 354 N134S; E138Q; P188S; P405A; Q418R; K510Y; ++++ A511T; 355 A2V;S107C; N134S; E138Q; P188S; T246A; ++ L332S; P405G; Q418V; K510D; A511T;356 N134S; E138Q; P188S; Q4181; S433Y; K510Y; +++ A511T; 357 N134S;E138Q; P188S; P405A; Q418V; K510S; ++++ A511T; 358 N134S; E138Q; P188S;T246A; P405L; Q418V; ++++ S433K; A511T; 359 N134S; E138Q; P188S; Q4181;K510S; A511T; +++ 360 S107C; N134S; E138Q; P188S; P405C; A505K; ++++K510Y; A511T; 361 N134S; E138Q; P188S; P405C; Q4181; A505K; ++++ K510D;A511T; 362 N134S; E138Q; P188S; L332S; P405F; Q4181; +++ K510D; A511T;363 N134S; E138Q; P188S; P405A; Q418V; A511T; ++++ 364 N134S; E138Q;P188S; P405L; Q418R; S433H; ++++ K510D; A511T; 365 N134S; E138Q; P188S;P405L; K510S; A511T; +++ 366 N134S; E138Q; P188S; S433H; K510D; A511T;+++ 367 N134S; E138Q; P188S; P405G; Q4181; S433N; ++++ K510Y; A511T; 368A2V; S107C; N134S; E138Q; P188S; L332S; ++ P405V; Q418R; A511T; 369N134S; E138Q; P188S; P405V; Q4181; S433N; ++++ K510S; A511T; 370 N134S;E138Q; P188S; P405V; Q418V; A511T; ++++ 371 S107L; N134S; E138Q; P188S;T246A; P405L; +++ Q418V; A505K; K510Y; A511T; 372 A2V; S107W; N134S;E138Q; P188S; P4050; + Q418R; A511T; 373 N134S; E138Q; P188S; T246A;S433K; K510D; +++ A511T; 374 N134S; E138Q; P188S; P405A; A511T; +++ 375A2V; G110D; N134S; E138Q; P188S; P405V; +++++ Q418V; A511T; 376 E87G;N134S; E138Q; P188S; P405V; A412V; +++++ Q4181; R508G; K509D; A511T; 377N134S; E138Q; P188S; A295V; P405V; Q418V; ++++ A511T; 378 N134S; E138Q;P188S; L209N; P405V; Q418V; +++++ L502S; R508H; K509H; A511T; 379 N134S;E138Q; P188S; T370A; P405V; Q418V; +++++ L502S; R508D; K509H; A511T; 380N134S; E138Q; P188S; P405V; Q418V; R508H; +++++ K509D; A511T; 381 N134S;E138Q; P188S; P405V; Q418V; R508H; +++++ K509H; A511T; 382 N134S; E138Q;P188S; P405V; Q418V; R508G; +++++ K509N; A511T; 383 N134S; E138Q; P188S;P405V; Q418V; A511T; +++++ 384 N134S; E138Q; P188S; P405W; Q418V; R508H;+++++ K509D;A511T; 385 H61R; N134S; E138Q; P188S; P405V; A416L; ++++Q418V; S433N; L502S; K509D; A511T; 386 N134S; E138Q; P188S; P405V;Q418V; K509D; +++++ A511T; 387 N134S; E138Q; P188S; A295T; P405V; Q418V;+++++ S458Q; R508H; K509D; A511T; 388 N134S; E138Q; P188S; P405V; Q418V;R508D; +++++ K509D; A511T; 389 N134S; E138Q; P188S; L209N; P405V; Q418V;+++++ A511T; 390 N134S; E138Q; P188S; P405V; Q418V; L502S; +++++ R508D;A511T; 391 N134S; E138Q; P188S; L209K; P405V; Q418V; +++++ S458Q; L502S;R508D; K509D; A511T; 392 N134S; E138Q; P188S; E303G; G401I; P405A; ++++Q418I; R508G; K509D; A511T; 393 N134S; E138Q; P188S;E303G; 04011; P405A;++++ Q418V; L502S; R508G; K509D; A511T; 394 E114G; N134S; E138Q; P188S;E3030; P405V; ++++ Q418V; L502S; R508G; K509D; A511T; 395 N134S; E138Q;P188S; E303G; G401S; P405A; ++++ A416L; Q418V; R508G; K509D; A511T; 396N134S; E138Q; P188S; G401S; P405A; Q418V; ++++ L502S; K509H; A511T; 397N134S; E138Q; P188S; P405V; A416L; Q418V; ++++ A511T; 398 N134S; E138Q;P188S; P405A; Q418V; S458Q; ++++ R508G; K509H; A511T; 399 N134S; E138Q;A162T; P188S; E303G; G401S; ++++ P405A; A416L; Q418I; L502S; R508G;K509Y; A511T; 400 N134S; E138Q; P188S; P405V; Q418V; S458Q; ++++ R508G;A511T; 401 N134S; E138Q; P188S; G401V; P405A; A412V; ++++ Q4181; L502S;R508G; K509H; A511T; 402 N134S; E138Q; P188S; E303G; G4011; P405A; ++++Q418V; L502S; K509H; A511T; 403 N134S; E138Q; P188S; E303G; P405V;A412V; ++++ Q418I; K509D; A511T; 404 N134S; E138Q; P188S; P405V; A416L;Q418V; ++++ L502S; R508G; K509H; A511T; 405 N134S; E138Q; P188S; E303G;P405V; Q418V; ++++ R508G; K509D; A511T; 406 N134S; E138Q; P188S; P405V;Q418V; L502S; ++++ R508G; K509D; A511T; 407 N134S; E138Q; P188S; G401S;P405L; A412V; ++++ A416L; Q418V; L502S; K509H; A511T; 408 N134S; E138Q;P188S; P405A; A412V; A416L; ++++ Q418V; R508G; K509D; A511T; 409 N134S;E138Q; P188S; P405V; Q418V; L502S; ++++ R508G; K509H; A511T; 410 N134S;E138Q; P188S; P405V; Q4181; R508G; ++++ K509D; A511T; 411 N134S; E138Q;P188S; G401V; P405V; Q418I; ++++ A505K; A511T; 412 N134S; E138Q; P188S;P405V; Q418V; A505K; ++++ A511T; 413 N134S; E138Q; P188S; E303G; G401L;P405V; ++++ Q418V; A505K; A511T; 414 N134S; E138Q; P188S; E303G; G401V;P405L; ++++ Q418I; A505K; A511T; 415 K22R; N134S; E138Q; P188S; E303G;G401V; ++++ P405A; Q418V; A511T; 416 N134S; E138Q; P188S; G264S; P405V;Q418V; ++++ Y500D; A505K; A511T; 417 N134S; E138Q; P188S; E303G; P405V;0418I; ++++ A511T; 418 N134S; E138Q; P188S; E303G; G401L; P405C; ++++Q418V; A511T; 419 N134S; E138Q; P188S; E303G; G401I; P405W; ++++ Q418V;A511T; 420 N134S; E138Q; P188S; E303G; G401L; P405V; ++++ Q4181; A505K;A511T; 421 N134S; E138Q; P188S; G401V; P405C; Q418V; ++++ A511T; 422N134S; E138Q; P188S; E303G; P405A; Q418V; ++++ A505K; A511T; 423 N134S;E138Q; P188S; P405V; Q418I; A511T; ++++ 424 N134S; E138Q; P188S; P405V;Q418V; D429E; ** S458Q; L502S; R508D; K509D; A511T; 425 N134S; E138Q;P188S; D212R; P405V; Q418V; ** S458Q; G487S; L502S; R508D; K509D; A511T;426 N134S; E138Q; P188S; S339G; P405V; Q418V; * S458Q; L502S; R508D;K509D; A511T; 427 N134S; E138Q; P188S; P405V; Q418V; N427K; * S458Q;L502S; R508D; K509D; A511T; 428 N134S; E138Q; P188S; P405V; Q418V;S458Q; ** V466Q; L502S; R508D; K509D; A511T; 429 N134R; E138Q; P188S;P405V; Q418V; S458Q; ** L502S; R508D; K509D; A511T; 430 T91R; N134S;E138Q; P188S; P405V; Q418V; * S458Q; L502S; R508D; K509D; A511T; 431G14V; N134S; E138Q; P188S; P405V; Q418V; *** S458Q; L502S; R508D; K509D;A511T; 432 L69E; N134S; E138Q; P188S; P405V; Q418V; ** S458Q; L502S;R508D; K509D; A511T; 433 N134S; E138Q; P188S; P405V; A409W; Q418V; *S458Q; L502S; R508D; K509D; A511T; 434 N134S; E138Q; P188S; S244H;P405V; Q418V; * S458Q; L502S; R508D; K509D; A511T; 435 H98P; N134S;E138Q; P188S; P405V; Q418V; * S458Q; L502S; R508D; K509D; A511T; 436N134S; E138Q; P188S; Q377K; P405V; Q418V; *** S458Q; L502S; R508D;K509D; A511T; 437 N134S; E138Q; P188S; A389V; P405V; Q418V; ** S458Q;L502S; R508D; K509D; A511T; 438 N134S; E138Q; P188S; P405V; Q418V;S433K; *** S458Q; L502S; R508D; K509D; A511T; 439 N134S; E138Q; P188S;P405V; Q418V; R432Q; * S458Q; L502S; R508D; K509D; A511T; 440 N134S;E138Q; P188S; Y380K; P405V; Q418V; * S458Q; L502S; R508D; K509D; A511T;441 L69Q; N134S; E138Q; P188S; P405V; Q418V; * S458Q; L502S; R508D;K509D; A511T; 442 N134S; E138Q; P188S; P405V; L406Y; Q418V; * S458Q;L502S; R508D; K509D; A511T; 443 N134S; E138Q; P188S; S283F; P405V;Q418V; * S458Q; L502S; R508D; K509D; A511T; 444 N134S; E138Q; P188S;P405V; G410N; Q418V; * S458Q; L502S; R508D; K509D; A511T; 445 N134S;E138Q; V153I; P188S; P405V; Q418V; ** S458Q; L502S; R508D; K509D; A511T;446 N134S; E138Q; P188S; T197P; P405V; Q418V; * S458Q; L502S; R508D;K509D; A511T; 447 N134S; E138Q; P188S; S244P; P405V; Q418V; *** S458Q;L502S; R508D; K509D; A511T; 448 N134S; E138Q; N177R; P188S; P405V;Q418V; * S458Q; L502S; R508D; K509D; A511T; 449 N134S; E138Q; P188S;S244G; P405V; Q418V; * S458Q; L502S; R508D; K509D; A511T; 450 N134S;E138Q ;P188S; V305I; P405V; Q418V; * R432C; S458Q; L502S; R508D; K509D;A511T; 451 R65Y; N134S; E138Q; P188S; P405V; Q418V; * S458Q; L502S;R508D; K509D; A511T; 452 R65G; N134S; E138Q; P188S; P405V; Q418V; *S458Q; L502S; R508D; K509D; A511T; 453 N134S; E138Q; P188S; P405V;A412M; Q418V; * S458Q; L502S; R508D; K509D; A511T; 454 N134S; E138Q;I186A; P188S; P405V; Q418V; * S458Q; L502S; R508D; K509D; A511T; 455N134S; E138Q; P188S; P405V; Q4181; S458Q; * L502S; R508D; K509D; A511T;456 N134S; E138Q; P188S; R342L; P405V; Q418V; * S458Q; L502S; R508D;K509D; A511T; 457 N134S; E138Q; P188S; G340P; P405V; Q418V; ** S458Q;L502S; R508D; K509D; A511T; 458 N134S; E138Q; P188S; P405V; Q418V;T436D; * S458Q; L502S; R508D; K509D; A511T; 459 N134S; E138Q; Q180H;P188S; P405V; Q418V; * S452N; S458Q; L502S; R508D; K509D; A511T; 460N134S; E138Q; P188S; S283E; P405V; Q418V; * S458Q; L502S; R508D; K509D;A511T; 461 N134S; E138Q; P188S; S306W; T370I; P405V; *** Q418V; S458Q;L502S; R508D; K509D; A511T; 462 N134S; E138Q; P188S; P405V; G410A;Q418V; * S458Q; L502S; R508D; K509D; A511T; 463 N134S; E138Q;P188S;P405V; G410R; Q418V; * S458Q; L502S; R508D; K509D; A511T; 464N134S; E138Q; P188S; P405V; A409Y; Q418V; * S458Q; L502S; R508D; K509D;A511T; 465 N134S; E138Q; P188S; P405V; Q418V; S458Q ** Y500R; L502S;R508D; K509D; A511T; 466 G14R; N134S; E138Q; P188S; P405V; Q418V; ***S458Q; L502S; R508D; K509D; A511T; 467 N134S; E138Q; P188S; V404A;P405V; Q418V; ** S458Q; L502S; R508D; K509D; A511T; 468 N134S; E138Q;P188S; P405V; Q418V; E421P; ** S458Q; L502S; R508D; K509D; A511T; 469N134S; E138Q; P188S; A389M; P405V; Q418V; ** S458Q; L502S; R508D; K509D;A511T; 470 N134S; E138Q; P188S; E227R; P405V; Q418V; ** S458Q; L502S;R508D; K509D; A511T; 471 N134S; E138Q; N174C; P188S; P405V; Q418V; **S458Q; L502S; R508D; K509D; A511T; 472 V104M; N134S; E138Q; P188S;P405V; Q418V; *** S458Q; L502S; R508D; K509D; A511T; 473 N134S; E138Q;P188S; G351C; P405V; Q418V; * S458Q; L502S; R508D; K509D; A511T; 474N134S; E138Q; P188S; Y380R; P405V; Q418V; * S458Q; L502S; R508D; K509D;A511T; 475 Q18I; N134S; E138Q; P188S; P405V; Q418V; * S458Q; L502S;R508D; K509D; A511T; 476 N134S; E138Q; P188S; P405V; Q418V; S458Q; **L499R; L502S; R508D; K509D; A511T; 477 N134S; E138Q; P188M; P405V;Q418V; S458Q; * L502S; R508D; K509D; A511T; 478 N134S; E138Q; N177T;P188S; P405V; Q418V; ** S458Q; L502S; R508D; K509D; A511T; 479 N134S;E138Q; P188S; P405V; Q418V; D429R; * S458Q; L502S; R508D; K509D; A511T;480 N134S; E138Q; P188S; P405V; Q418V; T436Q; * S458Q; L502S; R508D;K509D; A511T; 481 A63Y; N134S; E138Q; P188S; P405V; Q418V; * S458Q;L502S; R508D; K509D; A511T; 482 N134S; E138Q; P188S; P405V; Q418V;S452N; ** S458Q; L502S; R508D; K509D; A511T; 483 N134S; E138Q; P188S;P405V; G410H; Q418V; * S458Q; L502S; R508D; K509D; A511T; 484 N134S;E138Q; P188S; S266A; P405V; Q418V; * S458Q; L502S; R508D; K509D; A511T;485 V104I; N134S; E138Q; P188S; P405V; Q418V; *** S458Q; L502S; R508D;K509D; A511T; 486 N134S; E138Q; P188S; S283M; P405V; Q418V; *** S458Q;L502S; R508D; K509D; A511T; 487 N134S; E138Q; N177Q; P188S; P405V;G410C; ** Q418V; S458Q; L502S; R508D; K509D; A511T; 488 N134S; E138Q;P188S; A389I; P405V; Q418V; ** S458Q; L502S; R508D; K509D; A511T; 489N134S; E138Q; P188S; P405V; Q418V; S458Q; * V466R; L502S; R508D; K509D;A511T; 490 N134S; E138Q; P188S; P405V; Q418V; S452A; * S458Q; L502S;R508D; K509D; A511T; 491 N134S; E138Q; P188S; S244F; P405V; Q418V; *S458Q; L502S; R508D; K509D; A511T; 492 N134S; E138Q; P188S; P405V;A409V; Q418V; * S458Q; L479Q; L502S; R508D; 509D; A511T; 493 N134S;E138Q; P188S; P405V; Q418V; S458Q; ** V466E; L502S; R508D; K509D; A511T;494 N134S; E138Q; P188S; V318I; P405V; Q418V; ** S458Q; L502S; R508D;K509D; A511T; 495 N134S; E138Q; P188S; A389L; P405V; Q418V; ** S458Q;L502S; R508D; K509D; A511T; 496 N134S; E138Q; P188A; P405V; Q418V;S458Q; ** L502S; R508D; K509D; A511T; 497 N134S; E138Q; P188S; E205G;P405V; Q418V; ** S458Q; L502S; R508D; K509D; A511T; 498 N134S; E138Q;P188S; P405V; Q418V; T430H; ** S458Q; L502S; R508D; K509D; A511T; 499A63R; N134S; E138Q; P188S; P405V; Q418V; * S458Q; L502S; R508D; K509D;A511T; 500 N134S; E138Q; P188S; P405V; Q418V; S458Q; * L502S; R508D;K509D; A511K; 501 N134S; E138Q; P188S; P405V; Q418V; S458Q; ** Q474R;L502S; R508S; K509D; A511T; 502 N134S; E138Q; P188S; S306H; P405V;Q418V; * S458Q; L502S; R508D; K509D; A511T; 503 N134S; E138Q; P188S;D376P; P405V; Q418V; *** S458Q; L502S; R508D; K509D; A511T; 504 N134S;E138Q; P188S; Y380N; P405V; Q418V; * S458Q; L502S; R508D; K509D; A511T;505 N134S; E138Q; P188S; V398Y; P405V; Q418V; * S458Q; L502S; R508D;K509D; A511T; 506 G9D; N134S; E138Q; P188S; P405V; Q418V; * S458Q;G487T; Y500H; L502S; R508D; K509D; A511T; 507 N134S; E138Q; I186G;P188S; P405V; Q418V; ** S458Q; L502S; R508D; K509D; A511T; 508 N128H;N134S; E138Q; P188S; P405V; Q418V; * S458Q; L502S; R508D; K509D; A511T;509 N134S; E138Q; P188S; E227T; P405V; Q418V; * S458Q; L502S; R508D;K509D; A511T; 510 N134S; E138Q; P188S; V207L; P405V; Q418V; ** S458Q;L502S; R508D; K509D; A511T; 511 N134S; E138Q; P188S; L364I; P405V;Q418V; *** S458Q; L502S; R508D; K509D; A511T; 512 N134S; E138Q; P188S;S283T; P405V; Q418V; ** S458Q; L502S; R508D; K509D; A511T; 513 N134S;E138Q; P188S; S244A; P405V; M413R; ** Q418V; S458Q; L502S; R508D; K509D;A511T; 514 N134S; E138Q; P188S; P405V; Q418V; S458Q; * G487R; L502S;R508D; K509D; A511T; 515 N134S; E138Q; P188S; E227A; P405V; Q418V; *S458Q; L502S; R508D; K509D; A511T; 516 N134S; E138Q; P188S; V217L;P405V; Q418V; * S458Q; L502S; R508D; K509D; A511T; 517 N134S; E138Q;P188S; V399T; P405V; Q418V; * S458Q; L502S; R508D; K509D; A511T; 518H61R; N134S; E138Q; P188S; P405V; G410H; * Q418V; S458Q; L502S; R508D;K509D; A511T; 519 N134S; E138Q; P188S; P405V; Q418V; T430R; S458Q;L502S; R508D; K509D; A511T; ** 520 N134S; E138Q; P188S; M365N; P405V;Q418V; S458Q; L502S; R508D; K509D; A511T; ** 521 N134S; E138Q; P188I;P405V; Q418V; S458Q; L502S; R508D; K509D; A511T; ** 522 N134S; E138Q;I186Y; P188S; P405V; Q418V; S458Q; L502S; R508D; K509D; A511T; * 523N134S; E138Q; P188S; L226M; P405V; Q418V; S458Q; L502S; R508D; K509D;A511T; 524 N134S; E138Q; P188S; K224R; P405V; Q418V; * S458Q; L502S;R508D; K509D; A511T; 525 N134S; E138Q; P188S; E227H; P405V; Q418V; *S458Q; L502S; R508D; K509D; A511T; 526 N134S; E138Q; P188S; G401C;P405V; Q418V; * S458Q; L502S; R508D; K509D; A511T; 527 N134S; E138Q;P188S; P405V; Q418V; S458Q; ** Q474R; L502S; R508D; K509D; A511T; 528N134S; E138Q; P188S; Q341K; P405V; Q418V; # S433K; S458Q; L502S; R508D;K509D; A511T; 529 V104I; N134S; E138Q; P188S; L330V; P405V; # Q418V;S433K; S458Q; V466Q; L502S; R508D; K509D; A511T; 530 N134S; E138Q;P188S; S283T; P405V; Q418V; # S433K; S458Q; L502S; R508D; K509D; A511T;531 N134S; E138Q; P188S; S306H; P405V; Q418V; # S433K; S458Q; L502S;R508D; K509D; A511T; 532 R65G; N134S; E138Q; N174C; N177T; P188S; #K224R; G351C; P405V; Q418V; S433K; S458Q; L502S; R508D; K509D; A511T;533 R65G; N134S; E138Q; N174C; N177T; P188S; # K224R; V404A; P405V;Q418V; S433K; S458Q; L502S; R508D; K509D; A511T; 534 A63R; R65G; N134S;E138Q; P188S; K224R; # L226M; P405V; Q418V; S433K; S458Q; L502S; R508D;K509D; A511T; A512S; 535 Q18I; R65G; N134S; E138Q; P188S; K224R; #P405V; Q418V; S433K; S458Q; L502S; R508D; K509D; A511T; 536 A63R; R65G;N134S; E138Q; N174C; P188S; # L226M; P405V; Q418V; S433K; S458Q; L502S;R508D; K509D; A511T; 537 R65G; N128H; N134S; E138Q; N174C; P188S; #K224R; L226M; G351C; V404A; P405V; Q418V; S433K; S458Q; L502S; R508D;K509D; A511T; 538 A63R; R65G; N134S; E138Q; P188S; G351C; # V404A;P405V; Q418V; S433K; S458Q; G487R; L502S; R508D; K509D; A511T; 539N128H; N134S; E138Q; P188S; G351C; V404A; # P405V; Q418V; S433K; S458Q;G487R; L502S; R508D; K509D; A511T; 540 Q18I; N134S; E138Q; N174C; P188S;K224R; # L226M; G351C; P405V; Q418V; S433K; S458Q; L502S; R508D; K509D;A511T; 541 R65G; N128H; N134S; E138Q; N174C; N177T; # P188S; P405V;L406Y; Q418V; S433K; S458Q; L502S; R508D; K509D; A511T; 542 N128H;N134S; E138Q; N177T; P188S; L226M; # P405V; Q418V; S433K; S458Q; L502S;R508D; K509D; A511T; 543 Q18I; A63R; R65G; N134S; # E138Q; N174C; P188S;K224R; L226M; P405V; L406Y; Q418V; S433K; S458Q; L502S; R508D; K509D;A511T; 544 A63R; R65G; N134S; E138Q; N174C; N177T; # P188S; V404A;P405V; Q418V; S433K; S458Q; L502S; R508D; K509D; A511T; 545 N134S;E138Q; N174C; P188S; K224R; L226M; # E227R; G351C; V404A; P405V; Q418V;S433K; S458Q; L502S; R508D; K509D; A511T; 546 Q18I; R65G; N134S; E138Q;N177T; P188S; ## K224R; V404A; P405V; L406Y; Q418V; S433K; S458Q; L502S;R508D; K509D; A511T; 547 N128H; N1345; E138Q; N174C; P1885; L226M; ##G351C; P405V; Q418V; S433K; S458Q; L502S; R508D; K509D; A511T; 548 Q18I;A63R; N134S; E138Q; N174C; P188S; # G351C; P405V; Q418V; S433K; S458Q;G487R; L502S; R508D; K509D; A511T; 549 Q18I; R65G; N128H; N134S; E138Q;N174C; # N177T; P188S; K224R; P405V; Q418V; S433K; S458Q; L502S; R508D;K509D; A511T; 550 Q18I; N128H; N1345; E138Q; N174C; N177T; # P1885;P405V; Q418V; 5433K; S458Q; L502S; R508D; K509D; A511T; 551 Q18I; A63R;R65G; N134S; E138Q; P188S; # G351C; P405V; Q418V; S433K; S458Q; L502S;R508D; K509D; A511T; 552 N134S; E138Q; N174C; P1885; K224R; L226M; #G351C; P405V; L406Y; Q418V; S433K; S458Q; L5025; R508D; K509D; A511T;553 A63R; R65G; N134S; E138Q; N174C; P188S; ## K224R; L226M; P405V;Q418V; S433K; S458Q; L502S; R508D; K509D; A511T; 554 A63R; R65G; N134S;E138Q; N174C; N177T; ## P1883; L226M; P405V; L406Y; Q418V; S433K; S458Q;G487R; L502S; R508D; K509D; A511T; 555 Q18I; A63R; R65G; N128H; N134S;E138Q; ## P188S; P405V; Q418V; S433K; S458Q; G487R; L502S; R508D; K509D;A511T; 556 Q18I; N128H; N134S; E138Q; N174C; N177T; ## P188S; L226M;G351C; P405V; Q418V; S433K; S458Q; L502S; R508D; K509D; A511T; 557 R65G;N134S; E138Q; N174C; P188S; L226M; ## V404A; P405V; L406Y; Q418V; S433K;5458Q; G487R; L502S; R508D; K509D; A511T; 558 N134S; E138Q; P188S;V217L; P405V; Q418V; # S433K; S458Q; L502S; R508D; K509D; A511T; 559N134S; E138Q; P188S; A261D; S339G; Y380R; # P405V; G410R; Q418V; D429E;S433K; S458Q; Q474R; L502S; R508D; K509D; A511T; 560 N134S; E138Q;P188S; Y380N; P405V; G410A; # Q418V; S433K; S458Q; L502S; R508D; K509D;A511T; 561 N134S; E138Q; P188S; S266A; S339G; Y380N; # P405V; Q418V;S433K; S458Q; L502S; R508D; K509D; A511T; 562 N134S; E138Q; P188S;V217L; P405V; A409Y; # G410N; Q418V; S433K; S458Q; L502S; R508D; K509D;A511T; 563 N134S; E138Q; P188S; P405V; G410N; Q418V; # S433K; S458Q;L502S; R508D; K509D; A511T; 564 N134S; E138Q; P188S; P405V; A409W;G410A; # Q418V; S433K; S458Q; L502S; R508D; K509D; A511K; 565 N134S;E138Q; P188S; S339G; P405V; Q418V; # S433K; S458Q; L502S; R508D; K509D;A511T; 566 N134S; E138Q; P188S; P405V; A409W; G410R; # Q418V; S433K;S458Q; L502S; R508D; K509D; A511T; 567 L69E; N134S; E138Q; P188S; S266A;P405V; # Q418V; S433K; S458Q; L502S; R508D; K509D; A511T; 568 N134S;E138Q; P188S; S266A; P405V; Q418V; # S433K; S458Q; L502S; R508D; K509D;A511T; 569 L69E; N134S; E138Q; P188S; P405V; A409Y; # Q418V; D429E;S433K; S458Q; L502S; R508D; K509D; A511T; 570 L69E; N134S; E138Q; P188S;P405V; Q418V; # S433K; S458Q; L502S; R508D; K509D; A511T; 571 N134S;E138Q; P188S; S339G; P405V; G410A; # Q418V; D429E; S433K; S458Q; L502S;R508D; K509D; A511T; 572 N134S; E138Q; P188S; S339G; P405V; A409W; #G410R; Q418V; S433K; S458Q; L502S; R508D; K509D; A511T; 573 N134S;E138Q; P188S; P405V; G410A; Q418V; # S433K; S458Q; L502S; R508D; K509D;A511T; 574 N134S; E138Q; P188S; V217L; S266A; P405V; # Q418V; S433K;S458Q; L502S; R508D; K509D; A511T; 575 N134S; E138Q; P188S; V399T;G401C; P405V; # Q418V; R432Q; S433K; S458Q; L502S; R508D; K509D; A511T;576 N134S; E138Q; N177R; P188S; V399T; P405V; # Q418V; R432Q; S433K;S458Q; L502S; R508D; K509D; A511T; 577 N134S; E138Q; N177R; P188S;V399T; G401C; # P405V; Q418V; R432Q; S433K; S458Q; L502S; R508D; K509D;A511T; 578 N134S; E138Q; P188S; V399T; P405V; Q418V; # S433K; S458Q;L502S; R508D; K509D; A511T; 579 N134S; E138Q; P188S; V399T; G401C;P405V; # Q418V; S433K; S458Q; L502S; R508D; K509D; A511T; 580 N134S;E138Q; P188S; V398Y; G401C; P405V; # Q418V; S433K; S458Q; L502S; R508D;K509D; A511T; 581 N134S; E138Q; P188S; V398Y; V399T; G401C; # P405V;Q418V; R432Q; S433K; S458Q; L502S; R508D; K509D; A511T; 582 N134S;E138Q; N177R; P188S; V398Y; V399T; # P405V; Q418V; R432Q; S433K; S458Q;L502S; R508D; K509D; A511T; 583 N134S; E138Q; P188S; V398Y; V399T;P405V; # Q418V; R432Q; S433K; S458Q; L502S; R508D; K509D; A511T; 584N134S; E138Q; N177R; P188S; V398Y; P405V; # Q418V; S433K; S458Q; L502S;R508D; K509D; A511T; 585 N134S; E138Q; P188S; P405V; Q418V; R432Q; #S433K; S458Q; L502S; R508D; K509D; A511T; 586 N134S; E138Q; P188S;L364I; G401C; # P405V; Q418V; S433K; S458Q; L502S; R508D; K509D; A511T;587 N134S; E138Q; P188S; S244P; G401C; P405V; # Q418V; R432Q; S433K;S458Q; L502S; R508D; K509D; A511T; 588 N134S; E138Q; N177R; P188S;V398Y; P405V; # Q418V; R432Q; S433K; S458Q; L502S; R508D; K509D; A511T;589 N134S; E138Q; P188S; V398Y; P405V; Q418V; # S433K; S458Q; L502S;R508D; K509D; A511T; 590 N134S; E138Q; N177R; P188S; V398Y; G401C; #P405V; Q418V; R432Q; S433K; S458Q; L502S; R508D; K509D; A511T; 591N134S; E138Q; N177R; P188S; P405V; Q418V; # S433K; S458Q; L502S; R508D;K509D; A511T; 592 N134S; E138Q; P188S; A389M; P405V; Q418V; # S458Q;L502S; R508D; K509D; A511T; 593 N134S; E138Q; P188S; D376P; P405V;Q418V; # S458Q; L502S; R508D; K509D; A511T; 594 N134S; E138Q; P188S;A389I; P405V; Q418V; # S458Q; L502S; R508D; K509D; A511T; 595 N134S;E138Q; P188S; P405V; Q418V; S458Q; # Q474R; L502S; R508D; K509D; A511T;596 N134R; E138Q; P188S; S283F; P405V; Q418V; ## S458Q; L502S; R508D;K509D; A511T; 597 N134S; E138Q; P188S; S283M; P405V; Q418V; # S458Q;L502S; R508D; K509D; A511T; 598 N134S; E138Q; N177T; P188S; P405V;Q418V; # S458Q; L502S; R508D; K509D; A511T; 599 N134S; E138Q; I186G;P188S; P405V; Q418V; # S458Q; L502S; R508D; K509D; A511T; 600 N134S;E138Q; P188I; A389L; P405V; Q418V; # S458Q; L502S; R508D; K509D; A511T;601 N134S; E138Q; 174C; P188S; P405V; Q418V; # S458Q; Y500R; L502S;R508D; K509D; A511T; 602 V104I; N134S; E138Q; P188S; P405V; Q418V; #S458Q; L502S; R508D; K509D; A511T; 603 N134S; E138Q; P188I; P405V;Q418V; S458Q; # L502S; R508D; K509D; A511T; 604 N134S; E138Q; P188S;S244H; P405V; Q418V; # S458Q; L502S; R508D; K509D; A511T; 605 N134S;E138Q; P188S; M365N; P405V; Q418V; ## S458Q; L502S; R508D; K509D; A511T;606 P62S; N134S; E138Q; P188S; S244A; # P405V; L406Y; Q418V; S458Q;L502S; R508D; K509D; A511T; 607 N134S; E138Q; P188S; Q377K; P405V;Q418V; # S458Q; L502S; R508D; K509D; A511T; 608 N134S; E138Q; P188S;S283M; P405V; Q418V; # S458Q; Y500R; L502S; R508D; K509D; A511T; 609N134R; E138Q; P188S; V399T; P405V; Q418V; # S458Q; L502S; R508D; K509D;A511T; 610 N134S; E138Q; P188S; A389V; P405V; Q418V; # S458Q; L502S;R508D; K509D; A511T; 611 N134S; E138Q; P188S; D376P; P405V; Q418V; #S452G; S458Q; L502S; R508D; K509D; A511T; 612 N134S; E138Q; N177Q;P188S; P405V; Q418V; # S458Q; Y500R; L502S; R508D; K509D; A511T; 613N134R; E138Q; P188S; P405V; Q418V; S458Q; # L502S; R508D; K509D; A511T;614 N134S; E138Q; N177Q; P188S; 0377K; P405V; # Q418V; S458Q; L502S;R508D; K509D; A511T; 615 N134S; E138Q; P188S; P405V; Q418V; S452N; #S458Q; L502S; R508D; K509D; A511T; 616 N134S; E138Q; P188S; S244F;P405V; Q418V; # S458Q; Y500R; L502S; R508D; K509D; A511T; 617 Q6P;V104I; N134S; E138Q; P188S; P405V; # Q418V; S458Q; L502S; R508D; K509D;A511T; 618 N134S; E138Q; P188S; R403S; P405V; Q418V; # S458Q; Y500R;L502S; R508D; K509D; A511T; 619 N134S; E138Q; P188S; S283T; P405V;Q418V; # S458Q; L502S; R508D; K509D; A511T; 620 N134S; E138Q; P188S;L3641; P405V; Q418V; # S458Q; L502S; R508D; K509D; A511T; 621 N134S;E138Q; P188S; S283F; P405V; Q418V; # S458Q; L502S; R508D; K509D; A511T;622 A12T; N134R; E138Q; P188S; P405V; Q418V; # S458Q; L502S; R508D;K509D; A511T; 623 N134S; E138Q; P188S; P405V; Q418V; S458Q; # Y500R;L502S; R508D; K509D; A511T; 624 N134S; E138Q; P188S; P405V; G410N;Q418V; # S458Q; L502S; R508D; K509D; A511T; 625 N134S; E138Q; P188S;E227R; P405V; Q418V; # S458Q; L502S; R508D; K509D; A511T; 626 N134S;E138Q; P188S; P405V; Q418V; S458Q; # V466Q; L502S; R508D; K509D; A511T;627 N134S; E138Q; P188S; V318I; P405V; Q418V; # S458Q; V466E; L502S;R508D; K509D; A511T; 628 N134S; E138Q; P188S; S244P; P405V; Q418V; #S458Q; L502S; R508D; K509D; A511T; 629 N134S; E138Q; P188S; Y380K;P405V; Q418V; # S458Q; L502S; R508D; K509D; A511T; †Fatty alcohols forthe relative fatty alcohol measurements include: C14:0 (1-tetradecanol),C16:1 (cis Δ⁹-1-hexadecenol), C16:0 (1-hexadecanol), C18:1 (cisΔ¹¹-1-octadecenol), and C18:0 (1-octadecanol). + = 1.0 to 1.5 foldimprovement over wild-type M. algicola FAR at 30° C. ++ = 1.6 to 2.0fold improvement over wild-type M. algicola FAR at 30° C. +++ = 2.1 to3.0 fold improvement over wild-type M. algicola FAR at 30° C. ++++ = 3.1to 4.0 fold improvement over wild-type M. algicola FAR at 30° C. +++++= >4.1 fold improvement over wild-type M. algicola FAR at 30° C. * = 1.0to 1.5 fold improvement over Variant No. 391 at 37° C. ** = 1.6 to 2.0fold improvement over Variant No. 391 at 37° C. *** = ≧2.1 foldimprovement over Variant No. 391 at 37° C. # = 1.0 to 1.5 foldimprovement over Variant No. 438 at 40° C. ## = ≧1.6 fold improvementover Variant No. 438 at 40° C.

Example 3 Evaluation of M. algicola FAR Variants with Improved ActivityUsing Shake Flasks

A subset of FAR Maa Variant Nos. 1-423 were evaluated in shake flasks.Recombinant E. coli BW25113 strains (ΔfadE or ΔtorR knockouts)comprising a plasmid containing a heterologous gene encoding M. algicolaDG893 FAR variants were grown in 5 mL Luria Bertani (LB) or M9YE mediumsupplemented with 1% glucose and 30 μg/mL chloramphenicol (CAM), forapproximately 16-18 hours (overnight) in a shaker-incubator at 30° C.,200 rpm. A 5% inoculum was used to initiate fresh 50 mL culturecontaining M9YE medium supplemented with 30 μg/mL CAM and 0.4% glucose.The culture was incubated for 2 hours at 30° C., 200 rpm to an OD₆₀₀ of0.6-0.8, at which point expression of the heterologous FAR gene wasinduced with isopropyl-β-D-thiogalactoside (IPTG) (1 mM finalconcentration). Incubation was continued for about 24-48 hours under thesame conditions. An additional amount of glucose (0.5% w/v finalconcentration) was added to the culture at 3, 6, and 9 hours after IPTGinduction as needed. Samples were taken at various time points forextraction and analysis.

0.5 mL of cell culture was extracted with 1 mL of isopropanol:methylt-butyl ether (MTBE) (4:6 ratio) for 2 hours. The extract wascentrifuged, the upper organic phase transferred into a GC vial andanalyzed by the GC method described in Example 2. The typical shakeflask titers at 24 hours for those FAR Maa variants tested under theconditions described herein was in the range of 0.2 g/L to 1.5 g/L.Specifically, FAR Maa Variant No. 370 produced between 0.5 and 1 g/Lfatty alcohols and FAR Maa Variant No. 391 produced between 0.8 and 1.2g/L fatty alcohols.

Example 4 Chain Length Profile of Fatty Alcohols Exhibited byRecombinant E. coli Expressing Wild-type FAR Maa or FAR Maa Variants

The chain length profile of a subset of FAR Maa variants were evaluated.Table 3 provides the relative chain length distribution of fattyalcohols exhibited by recombinant E. coli strains expressing wild-typeFAR Maa or FAR Maa variants.

TABLE 3 Relative chain length distribution of fatty alcohols exhibitedby recombinant E. coli strains expressing wild-type FAR Maa or FAR Maavariants Relative Chain Length Distribution of Temperature FAR Variantfatty alcohols^(a) E. coli strain (° C.) No. C14:0 C16:1 C16:0 C18:1C18:0 BW25113-ΔfadE 30 Wild-type 8 30 30 32 <1 BW25113-ΔtorR ″ ″ 10 3030 30 <1 ″ ″ 370 10 42 29 19 <1 ″ ″ 391 10 40 29 21 ″ ″ ″ 436 19 46 2213 ″ ″ ″ 438 21 48 20 11 ″ ″ ″ 547 18 45 24 13 ″ ″ ″ 555 27 42 20 11 ″ ″″ 556 21 45 24 13 ″ 37 436 17 37 34 12 ″ ″ ″ 438 20 40 30 10 ″ ″ ″ 54726 43 25 6 ″ ″ ″ 555 28 42 24 6 ″ ″ ″ 556 28 43 23 6 ″ ″ 40 547 25 38 307 ″ ″ ″ 555 23 37 31 9 ″ ″ ″ 556 28 38 28 6 ″ MG1655-7740 30 436 31 3327 9 ″ ″ ″ 438 35 34 24 7 ″ ″ 37 436 32 23 38 7 ″ ″ ″ 438 36 24 34 6 ″W3110-10-ΔfhuA 30 436 27 39 26 8 ″ ″ ″ 438 30 41 23 6 ″ ″ 37 436 38 3126 5 ″ ″ ″ 438 39 31 25 5 ″ ^(a)The relative chain length distributionis expressed as a % of the total fatty alcohols detected via GC-FID.Fatty alcohols include: C14:0 (1-tetradecanol), C16:1 (cisΔ⁹-1-hexadecenol), C16:0 (1-hexadecanol), C18:1 (cis Δ¹¹-1-octadecenol),and C18:0 (1-octadecanol). No C14:1 (1-tetradecanol) was detected.

Example 5 Evaluation of M. algicola FAR Variants with Improved ActivityUsing Fermentors

A. Evaluation of FAR Maa Variants Using a Fed-batch DO-Stat FermentationProcess

In an aerated, agitated stirred tank 10 L fermentor, 3.0 L of growthmedium containing 33.85 g 5×M9 powder (BD Difco), 6 g Bacto yeastextract (BD), 3 g ammonium phosphate dibasic (Sigma-Aldrich), 15 gammonium sulfate (EMD), 9 g glucose (Sigma), 1.48 g magnesium sulfate,heptahydrate (Sigma), 44 mg Calcium chloride, Dihydrate (Sigma), 15 mltrace elements solution, 12.6 mg EDTA (Sigma-Aldrich), 150 mg Fe(III)Citrate (Sigma), 6.75 mg Thiamine.HCl (Sigma), and 90 μgchloroamphenicol (Sigma Chemical Co.) was brought to a temperature of30° C. The fermentor was inoculated with a late exponential culture ofE. coli strain containing M. algicola DG893 FAR (FAR Maa) Variant Nos.370 and 391 to a starting optical density (OD₆₀₀) of about 1.0. Theinoculum was grown in a 1000 mL baffled shake flask containing 200 ml of47.6 g/L terrific broth powder (Difco), 4 ml/L glycerol (Sigma), and3011g/ml chloroamphenicol (Sigma Chemical Co.) at 30° C., 250 rpm untilthe OD600 reached ˜8.0-10.0. The fermentor was agitated at 300-1200 rpmand air supplied at 3.0 L/min to maintain a minimum dissolved oxygenlevel of 30% of saturation. The pH of the culture was controlled at 7.0by addition of 5 N sodium hydroxide.

After consumption of the 3 g/L initial glucose, an exponential fed-batchgrowth phase with a specific growth rate of 0.22 h⁻¹ was initiated byexponential addition of feed solution containing 500 g/L glucose(Sigma), 13.06 g/L magnesium sulfate, heptahydrate (Sigma), 100 g/Lammonium sulfate (EMD), 10 ml/L trace elements solution, 8.4 mg/L EDTA(Sigma-Aldrich), 100 mg/L Fe(III) Citrate (Sigma), 4.5 mg/L Thiamine.HCl(Sigma), and 30 μg/L chloroamphenicol (Sigma Chemical Co.) to fermentor.After 16 hours of fed-batch culture and 1 hour delay, the expression ofFAR Maa Variant Nos. 370 and 391 was induced by the addition ofisopropyl-β-D-thiogalactoside (IPTG) (US Biological) to a finalconcentration of 1 mM. Production of fatty alcohol was maintained byaddition of a feed solution containing 650 g/L glucose (Sigma), 5.6 g/Lmagnesium sulphate, heptahydrate (Sigma), 6.5 g/L ammonium sulfate(EMD), 15 ml/L trace elements solution, 6.3 mg/L EDTA (Sigma-Aldrich),75 mg/L Fe(III) Citrate (Sigma), 5.6 mg/L Thiamine.HCl (Sigma), 30 μg/Lchloroamphenicol (Sigma Chemical Co.), and 1 mM IPTG (US Biological).The addition of feed solution (10 g/L glucose per pulse) was triggeredafter a 45 minutes delay following dissolved oxygen (DO) spikes above40%. The culture was grown for another 120 hours at 30° C. Samples weretaken at various time points for extraction and analysis. Extraction andquantification of fatty alcohols were performed as described in Examples2 and 3.

Under the conditions tested, the total production of fatty alcohols was˜45-50 g/L for E. coli BW25113 ΔtorR expressing FAR Maa Variant Nos. 370and 391 as compared to 12-15 g/L for the same strain expressing thewild-type FAR Maa of SEQ ID NO: 2 under the fermentation conditionsreported in WO 2011/100835.

B. Evaluation of FAR Maa Variants at Different Temperatures Using aShort-batch Fermentation Process

A short-batch fermentation process was used to more efficiently compareimproved FAR Maa variants for relative FOH activity in the presence ofexcess glucose and under controlled bioreactor conditions. In anaerated, agitated stirred tank 10 L fermentor, 3.0 L of growth mediumcontaining 33.85 g 5x M9 powder (BD Difco), 6 g Bacto yeast extract(BD), 3 g ammonium phosphate dibasic (Sigma-Aldrich), 15 g ammoniumsulfate (EMD), 9 g glucose (Sigma), 1.48 g magnesium sulfate,heptahydrate (Sigma), 44 mg calcium chloride dehydrate (Sigma), 15 mltrace elements solution, 12.6 mg EDTA (Sigma-Aldrich), 150 mg Fe(III)citrate (Sigma), 6.75 mg thiamine.HCl (Sigma), and 90 μgchloroamphenicol (Sigma Chemical Co.) was brought to a temperature of30° C. The fermentor was inoculated with a late exponential culture ofE. coli strain containing M. algicola DG893 FAR (FAR Maa) Variant Nos.370 and 438 to a starting optical density (OD₆₀₀) of about 1.0. Theinoculum was grown in a 1000 mL baffled shake flask containing 200 ml of47.6 g/L terrific broth powder (Difco), 4 ml/L glycerol (Sigma), and 30μg/ml chloroamphenicol (Sigma Chemical Co.) at 30° C., 250 rpm until theOD₆₀₀ reached ˜8.0-10.0. The fermentor was agitated at 300-1200 rpm andair supplied at 3.0 L/min to maintain a minimum dissolved oxygen (DO)level of 30% of saturation. The pH of the culture was controlled at 7.0by addition of 5 N sodium hydroxide.

After consumption of the 3 g/L initial glucose, an exponential fed-batchgrowth phase with a specific growth rate of 0.22 h⁻¹ was initiated byexponential addition of feed solution containing 500 g/L glucose(Sigma), 13.06 g/L magnesium sulfate, heptahydrate (Sigma), 100 g/Lammonium sulfate (EMD), 10 ml/L trace elements solution, 8.4 mg/L EDTA(Sigma-Aldrich), 100 mg/L Fe(III) citrate (Sigma), 4.6 mg/L thiamine.HCl(Sigma), and 30 μg/L chloroamphenicol (Sigma Chemical Co.) to thefermentor.

After 16 hours, 0.75-1 L of fed-batch culture was harvested andtransferred to a 10 L fermentor containing 3 L production medium (45.13g 5×M9 powder (BD Difco), 300 g glucose (Sigma), 1.497 g magnesiumsulfate, heptahydrate (Sigma), 58.7 mg calcium chloride dehydrate(Sigma), 20 ml trace elements solution, 16.8 mg EDTA (Sigma-Aldrich),200 mg Fe(III) citrate (Sigma), 9 mg thiamine.HCl (Sigma), and 120 μgchloroamphenicol (Sigma Chemical Co.). Deionized water (DIW) was used toadjust the total initial working volume to 4.0 L and the starting OD₆₀₀to 20. The fatty alcohol production was initiated by induction of theexpression of FAR Maa Variant Nos. 370 and 438 via addition ofisopropyl-6-D-thiogalactoside (IPTG) (US Biological) to a finalconcentration of 1 mM. The fermentor was agitated at 300-1200 rpm andair supplied at 3.0 L/min to maintain a minimum dissolved oxygen (DO)level of 30% of saturation. The pH of the culture was controlled at 7.0by addition of 5 N sodium hydroxide. The production was carried on atthe desired temperature (i.e., 30, 34, or 37° C.) for ˜30-48 hours.Samples were taken at various time points for extraction and analysis.Extraction and quantification of fatty alcohols were performed asdescribed in Examples 2 and 3.

Under the conditions tested, the total production of fatty alcohols at˜30 hours for Variant No. 370 was 5.6, 3.9, and 1.9 g/L at 30° C., 34°C., and 37° C., respectively. The total production of fatty alcohols at˜30 hours for Variant No. 438 was 7.2, 8.3, and 6.7 g/L at 30° C., 34°C., and 37° C., respectively.

Example 6 Wild-type M. aquaeolei FAR Gene Acquisition and VectorConstruction

Gene acquisition of wild-type M. aquaeolei FAR (“FAR Maq”) is describedin the published application WO 2011/008535. The amino acid sequence ofM. aquaeolei FAR can be found at GenBank Accession Number YP_(—)959486,and is designated SEQ ID NO:5. The polynucleotide sequence of thecodon-optimized gene encoding the FAR polypeptide of SEQ ID NO:5 isdesignated SEQ ID NO:4. The M. aquaeolei FAR gene and genes encodingvariants of the M. aquaeolei FAR were cloned into the vector pCK110900(depicted as FIG. 3 in US Pat. Appln. Pub. 20060195947) under thecontrol of a lac promoter as described in WO/2011/008535. The resultingplasmids were introduced into E. coli BW25113 ΔtorR (Baba et al.,Molecular Systems Biology, 2006 doi:10,1038/msb4100050 Article No.2006.0008) by routine transformation methods.

Example 7 Evaluation of Wild-type M. aquaeolei FAR in Shake Flask

Recombinant E. coli BW25113 ΔtorR strain comprising a plasmid containinga heterologous gene encoding M. aquaeolei FAR was grown in 5 mL M9YEmedia supplemented with 1% glucose and 30 μg/mL chloramphenicol (CAM),for approximately 16-18 hours (overnight) in a shaker-incubator at 30°C. at 200 rpm. A 5% inoculum was used to initiate fresh 50 mL cultureusing M9YE medium supplemented with 30 μg/mL CAM and 0.4% glucose. Theculture was incubated for 2 hours at 30° C., 200 rpm to an OD₆₀₀ of0.6-0.8, at which point expression of the heterologous FAR gene wasinduced with isopropyl-β-D-thiogalactoside (IPTG) (1 mM finalconcentration). An additional amount of glucose (0.5% w/v final conc.)was added to the culture at 3 and 6 hours after IPTG induction.Incubation was continued for about 24 hours under the same conditions.

0.5 mL of cell culture was extracted with 1 mL of isopropanol:methylt-butyl ether (MTBE) (4:6 ratio) for 2 hours. The extract wascentrifuged and the upper organic phase was transferred into a GC vialand analyzed by the following GC-FID method using DB-5MS column (length30 m, I.D. 0.32 mm, film 0.25 urn): start temp. 150° C., increase thetemperature at a rate of 25° C./min to 246° C. and hold for 1.81 min.Total run time, 5.65 min. Under the above GC conditions the approximateretention times (min) of produced fatty alcohols and acids were asfollows: 3.19, C14:0-OH; 3.48, C14:0-OOH; 3.91, C16:1-OH; 3.98,C16:0-OH; 4.15, C16:0-OOMe (internal standard); 4.21, C16:1-OOH; 4.28,C16:0-OOH; 4.83, C18:1-OH; 4.92, C18:0-OH; 5.31, C18:0-OOH and 5.51,C18:1-OOH. Identification of individual fatty alcohol was done bycomparison to commercial standards (Sigma Chemical Company, 6050 SpruceSt. Louis, Mo. 63103). Under the conditions tested, typical shake flasktiters at 24 hours for the wild-type M. aquaeolei FAR was ˜0.2-0.25 g/L.Fatty alcohols include: 2% 014:0-OH (1-tetradecanol), 8% C16:1-OH (cisΔ9-1-hexadecenol), 39% 016:0-OH (1-hexadecanol), 49% 018:1-OH (cisΔ11-1-octadecenol), and <1% 018:0-OH (1-octadecanol).

Example 8 Evaluation of M. aquaeolei FAR Variants with Improved ActivityUsing Microtiter Plates

FAR Maq variants were grown in 96-well shallow plates containing 180 μLM9YE medium supplemented with 1% glucose and 30 μg/mL chloramphenicol(CAM), for approximately 16-18 hours (overnight) in a shaker-incubatorat 30° C., 200 rpm. A 5% inoculum was used in 96-deep-well plates toinitiate fresh 380 μL M9YE medium culture supplemented with 30 μg/mL CAMand 0.5% glucose. The culture was incubated for 2 hours at 30° C., 250rpm to an OD₆₀₀ of 0.6-0.8, at which point expression of theheterologous FAR gene was induced with isopropyl-β-D-thiogalactoside(IPTG) (1 mM final concentration). Incubation was continued for about 24hours under the same conditions. An additional amount of glucose (0.5%w/v final conc.) was added to the culture at 6 hours after induction byIPTG. Cell cultures were extracted with 1 mL of isopropanol:methylt-butyl ether (MTBE) (4:6 ratio) for 2 hours. The extracts werecentrifuged and the upper organic phase was transferred intopolypropylene 96-well plates and analyzed by the GC-FID method describedin Example 7. Identification of individual fatty alcohol was done bycomparison to commercial standards (Sigma Chemical Company, 6050 SpruceSt. Louis, Mo. 63103).

Table 4 provides the relative fatty alcohol production for illustrativevariants relative to wild-type M. aquaeolei FAR. Codon-optimized SEQ IDNO:4 was mutated and used to express FAR Maq Variant Nos. 1-89. Relativefatty alcohol production is presented as fold improvement over wild-typeFAR Maq at 30° C. In Table 4, the amino acid substitutions listed foreach variant correspond to residue positions of SEQ ID NO:5 (e.g.,“G402A” means that the residue at position 402 in SEQ ID NO:5 (glycine)is substituted with alanine), and the amino acid positions weredetermined by optimal alignment with SEQ ID NO:5.

TABLE 4 Variant FAR polypeptides and total relative production of fattyalcohols Relative fold improvement in Amino acid substitutions relativeto fatty alcohol Variant No. SEQ ID NO: 5 production† 1 G402A; + 2N135K; ++ 3 E238L; + 4 A74K; ++ 5 A2T; G113A; ++ 6 D141C; + 7 Y501N; ++8 D430K; ++ 9 Y501Q; + 10 S502R; + 11 K230R; Y501G; + 12 A2H; E72Q; ++13 E228G; ++ 14 S434K; Y501P; ++ 15 S502G; A512K; +++ 16 Y381R; + 17A2F; + 18 E139Q; + 19 L503R; ++ 20 S434K; ++ 21 1438V; A512G; ++ 22 A2T;D116E; + 23 Q508G; + 24 A108L; D430K; ++ 25 A2F; H8N; + 26 A45V; A374V;K511D; + 27 A512P; ++ 28 L503Q; ++ 29 A512S; ++ 30 A512K; +++ 31 A2Q; ++32 N135K; R412H; + 33 A2H; D116A; ++ 34 Q508S; R509A; K510G; K511C;A512S; ++ A513L; 35 D411R; S434K; + 36 A2F; H8N; A108C; + 37 A2H; P63S;G113A; D141C; ++ 38 A2G; + 39 S434F; + 40 A2P; A108C; + 41 A2Q; G113A;D116A; ++ 42 L503R; R509D; ++ 43 K511D; + 44 Y501G; ++ 45 A512R; ++ 46A375Y; + 47 P406S; K511D; + 48 E72Q; Q508G; + 49 A512G; +++ 50 S434W; +51 T505R; + 52 K511G; ++ 53 A2H; G113A; ++ 54 S502G; ++ 55 N459Q; L503Q;++ 56 Y501S; + 57 A512Q; +++ 58 A2P; + 59 A512T; +++ 60 Y501W; + 61G111S; Y501P; + 62 A2T; ++ 63 Y501P; ++ 64 L503S; ++ 65 A74L; ++ 66P63Q; + 67 E205R; + 68 R66N; ++ 69 D422A; + 70 S77G; ++ 71 E88Q; + 72H8K; + 73 E238C; + 74 D199G; + 75 Q5F; + 76 A513Y; + 77 A9L; + 78 N459G;++ 79 E72S; + 80 E205G; + 81 A375Q; + 82 Q181D; ++ 83 Q4I; + 84 E497Y;++ 85 A108R; ++ 86 Q5N; ++ 87 E497F; + 88 T505K; + 89 D141G; + †Fattyalcohols for the relative fatty alcohol measurements include: C14:0(1-tetradecanol), C16:1 (cis Δ⁹-1-hexadecenol), C16:0 (1-hexadecanol),C18:1 (cis Δ¹¹-1-octadecenol), and C18:0 (1-octadecanol). + = 1.0 to 1.5fold improvement over wild-type M. aquaeolei FAR at 30° C. ++ = 1.6 to2.0 fold improvement over wild-type M. aquaeolei FAR at 30° C. +++ =≧2.1 fold improvement over wild-type M. aquaeolei FAR at 30° C.

Table 4A illustrates that many substitutions in SEQ ID NO:5 (“Maq”)which improve fatty alcohol yield correspond to beneficial substitutionsin SEQ ID NO:2 (“Maa”). “FIOP”, or “Fold Improvement Over Parent”, isthe average observed fold-improvement in fatty alcohol production in E.coli expressing a FAR Maq variant relative to E. coli expressing thewild-type M. aquaeolei FAR.

TABLE 4A Maq FIOP Maa A2G + A2G A2T + A2T A2P + A2P A2F + A2F A2Q + A2QA74K + A73K N135K + N134K E139Q + E138Q D141C + D140C E205G + E204GE228G + E227G E238L + E237L A375Y + A374Y Y381R + Y380R G402A + G401AD430K + D429K S434W + S433W S434F + S433F S434K + S433K N459G + S458GY501N + Y500N Y501S + Y500S Y501Q + Y500Q Y501G + Y500G Y501P + Y500PY501W + Y500W S502G + S501G S502R + S501R L503Q + L502Q L503S + L502SL503R + L502R T505R + A504R Q508G + T507G Q508R + T507R K510D + K509DK511D + K510D K511P + K510P K511G + K510G A512S + A511S A512G + A511GA512R + A511R A512K + A511K A512P + A511P A512Q + A511Q A512T + A511T += ≧1.1 fold improvement over wild-type M. aquaeolei FAR

Example 9 Substitutions in M. aquaeolei FAR

Table 5 is a listing of substitutions that may be introduced into SEQ IDNO:5, or a polypeptide having at least 70% identity to SEQ ID NO:5 asdescribed elsewhere herein, to produce FAR variants. The substitutionsand substitution sets are numbered with reference to SEQ NO:5.

As demonstrated in Example 8, numerous substitutions in SEQ ID NO:5 atpositions corresponding to beneficial substitutions in SEQ ID NO:2 wereshown to result in FARs that support increased fatty alcohol production.Table 5, below, provides contemplated FAR variants of the invention. InTable 5, positions, sets of positions, substitutions and substitutionsets are provided with reference to SEQ ID NO:5.

TABLE 5 Variant No. Corresponding substitutions in FAR Maq (SEQ ID NO:5) 1 D411S 2 A284V 3 D199Q 4 A284K 5 P340V 6 N459G 7 V464T 8 D411R 9E289Q 10 D411Q 11 E139Q 12 A284M 13 R237K 14 L34V 15 L414L 16 I288L 17L417V 18 R118D 19 E139L, A145Q 20 E360L, A512T 21 N135R 22 L210N, V413V23 S245G 24 E84R 25 V413V 26 E238L 27 L210N 28 T102L 29 R152L 30 V464E31 I438V, A512T 32 E205G 33 R66Q 34 G341V 35 N459M 36 A444T 37 L258K 38A375K 39 I43L 40 P340G 41 N135K 42 L398L 43 N459Q 44 A488Y 45 G341S,P406S 46 V413F 47 V413C 48 D116A 49 D422R 50 P406S 51 D141C 52 P406F 53K15W 54 D430Q 55 S434K 56 A9K 57 P406G 58 S502G 59 K230R 60 H378C 61Y381R 62 K510S 63 N419I 64 R507S 65 K511G 66 Y501C 67 E497A 68 A2H 69P406C 70 A506K 71 D10F 72 T307W 73 E304G, T431I 74 T505G 75 N419V 76Y501P 77 R507G 78 D422Y 79 S434H 80 K511P 81 L503Q 82 A512P 83 A512G 84L503A 85 S332V 86 N419R 87 A2W 88 D422I 89 A2D 90 K225R 91 N419Y 92 G402V 93 L503R 94 K511Y 95 L503S 96 Y501L 97 T505R 98 E138L 99 S434W 100Q508G 101 Q181R, T247A 102 G402L 103 T122S, S434L 104 K511S 105 Q508A106 S77K 107 K510H 108 K511D 109 A512R 110 G402S 111 A108L 112 R509G 113H8N 114 Y501W 115 H378Y 116 S401L 117 V500P 118 T75K 119 Q508R 120 E304G121 A512T 122 Q114L, D422P 123 A2T, L333S 124 I3R 125 V500A 126 A2F 127A512S 128 A512K 129 Q114D 130 S502R 131 L365F, S401A 132 A375Y 133 Y501G134 A512Q 135 D422S 136 K15N 137 K510E 138 T122H 139 D430K 140 A512I 141A2V 142 Y501N 143 S434F 144 V500H 145 T437K 146 A2V, A108C 147 A2G,L149E 148 E206P, A513T 149 L417L 150 S385R 151 A2Q 152 K499A, L503R 153Y501Q 154 Y501S 155 P406L 156 Q508S 157 V500S 158 T307F 159 L227A 160G18D 161 A9S 162 K510G 163 A74K 164 K511A 165 S434N 166 Q5S, P406S 167L503P 168 A2P 169 D422L 170 V500R 171 D422V 172 D422N 173 A51V, A512T174 R382C, A512T 175 A143V, A512T 176 P189S, A512T 177 T75P 178 A473V,A512T 179 V186A, A512T 180 P445S, A512T 181 R460H, D465G, V500P, A512T182 V78A, A512T 183 K261T, A512T 184 H99R, A512T 185 K23E, A512T 186V25I, R404C, A512T 187 A126V, A512T 188 A300T, A512T 189 R221C, A512T190 V186A, A334T, A512T 191 N459L, A512T 192 I112S, A512T 193 E72K,N459L, A512T 194 L94V 195 Y447H, A512T 196 D246N, A512T 197 I329T, A512T198 N264P, D411S, A512T 199 N491S, A512T 200 E139Q, P189S, E228G, E238L201 N135R, E139Q, P189S 202 E139Q, P189S 203 A121V, N135K, N459Q, A512T204 Q4R, T11T, E139Q 205 A92I, N135R, P189S, K261R, A512T 206 N135K,E139Q, P189S, N459Q 207 N459Q, I485V 208 N135K, E228G 209 N135S, E139Q,P189S 210 G113A, N135K, P189S 211 N135S, E139Q, P189S, A512T 212 N135K,P189S 213 S13V, N459Q, A512T 214 N135R, D411Q, V413C, N459Q 215 G103C,N135R, E139Q, P189S 216 N135S, E139Q, E206G, A512T 217 N135S, E139Q,P189S, F441L, A512T 218 N135R, E203G, K214R 219 N135R, P189S, K214R,K261R, I438V, N459Q 220 N135S, P189S, D411Q, V413V, N459Q, A512T 221N135R, P189S 222 L55P, A367T, D411S, N459Q, A512T 223 N135S, E228G 224A51S, N135K, E139Q, N459Q 225 N459Q, A512T 226 N135S, E139Q, P189S,E228G 227 E139Q, I270T, A512T 228 D397G, A512T 229 D116H, N135R, P189S230 N135R, E139Q, N161S, P189S, E304G 231 R61H, G113A, N135R, E228G,V291I, D411Q, I438V, N459Q 232 E139Q, G351S, A512T 233 P189S, E228G 234E139Q, P189S, T307N 235 N135K, P189S, N459Q 236 N135K, N459L 237 S133G,N459Q, A512T 238 A2G, N135S, E139Q, P189S, A512T 239 A2I, G113A, N135K,E139Q, P189S, A512T 240 N135S, E139Q, P189S, E228G, A512T 241 G113A,N135K, E139Q, P189S, A512T 242 A2G, G113A, N135R, E139Q, P189S, K511P,A512R 243 A2H, G113A, N135S, E139Q, P189S, N459Q, A512T 244 A2D, G113A,N135R, E139Q, P189S, D422V, K510H, A512Q, A513T 245 N135S, E139Q, P189S,V208I, K511P, A512G 246 N135S, E139Q, P189S, N459Q, K511P, A512K 247E123R, N135S, E139Q, P189S, K511P, A512R 248 N135S, E139Q, P189S, N459Q,K511P, A512R 249 N135S, E139Q, P189S, K511P, A512R, A513T 250 N135S,E139Q, P189S, E228G, N459Q, K511P, A512K 251 A2G, G113A, N135K, E139Q,P189S, N459Q, K510H, K511P, A512Q 252 N135S, E139Q, P189S, D422S, N459Q,K510H, K511P, A512R 253 G113A, N135R, E139Q, P189S, D422R, N459Q, K511P,A512S 254 N135S, E139Q, P189S, K511P, A512G, A513T 255 A2H, N135S,E139Q, P189S, K511P, A512S, A513T 256 A2W, G113A, N135R, E139Q, P189S,K510H, K511P, A512G 257 A2N, N135S, E139Q, P189S, D422V, A512T 258N135S, E139Q, P189S, D422R, N459Q, K511P, A512S, A513T 259 N135S, E139Q,P189S, D422L, K510H, K511P, A512K 260 A2P, N135S, E139Q, P189S, K511P,A512R, A513T 261 N135K, E139Q, P189S, A512T 262 G113A, N135S, E139Q,P189S, A512T 263 N135S, E139Q, P189S, N459Q, A512T, A513T 264 A2G, A74V,G113A, N135S, E139Q, P189S, E228G, A512T 265 E139Q, P189S, A512T, A513T266 N135R, E139Q, P189S, A512T 267 N135S, E139Q, P189S, D422L, N459Q,A512T 268 N135S, E139Q, P189S, N459Q, A512T 269 N135S, E139Q, P189S,D422R, N459Q, A512T 270 G113A, E139Q, P189S, A512T 271 N135S, E139Q,P189S, E228G, A512T, A513T 272 G113A, N135S, E139Q, P189S, E206P, D422R,A512T 273 A2G, G113A, E139Q, P189S, A512T 274 N135S, E139Q, P189S,A512T, A513T 275 N135S, E139Q, P189S, D430N, V500S, K510G, A512T 276N135S, E139Q, P189S, V413V, D430K, V500H, K510E, A512T 277 N135S, E139Q,P189S, D430N, K510N, A512T 278 N135S, E139Q, P189S, V413V, D430K, A512T279 N135S, E139Q, P189S, V413V, D430K, V500N, A512T 280 N135S, E139Q,P189S, V500R, K510R, A512T 281 N135S, E139Q, P189S, D430E, V500N, K510G,A512T 282 N135S, E139Q, Q181R, P189S, T307W, V500R, K510G, A512T 283T122S, N135S, E139Q, P189S, T307W, A375Y, V500R, K510S, A512T 284 N135S,E139Q, P189S, D430K, V500N, T505G, K510G, A512T 285 V78I, N135S, E139Q,P189S, T307W, A512T 286 N135S, E139Q, P189S, D430N, V500R, T505G, K510G,A512T 287 N135S, E139Q, P189S, D430E, V500N, A512T 288 N135S, E139Q,P189S, V413V, D430E, V500I, K510D, A512T 289 N135S, E139Q, P189S, T307W,V413V, D430K, V500S, A512T 290 N135S, E139Q, P189S, D430N, V500R, K510S,A512T 291 N135S, E139Q, P189S, V413V, V500I, K510G, A512T 292 N135S,E139Q, P189S, D430E, V500H, K510G, A512T 293 N135S, E139Q, P189S, T505R,K510N, A512T 294 N135S, E139Q, P189S, V413V, D430E, V500R, K510G, A512T295 N135S, E139Q, P189S, D430N, V500I, K510G, A512T 296 N135S, E139Q,P189S, T307W, A375Y, V500R, T505R, A512T 297 N135S, E139Q, P189S, D430E,K510D, A512T 298 N135S, E139Q, Q181R, P189S, T307W, V500H, K510S, A512T299 N135S, E139Q, P189S, L398I, A512T 300 D10F, N135S, E139Q, P189S,L503Q, Q508A, A512T 301 N135S, E139Q, P189S, G402V, L503S, A512T 302N135S, E139Q, P189S, G402S, A512T 303 D10F, Q114L, N135S, E139Q, P189S,G402L, L503S, Q508A, A512T 304 N135S, E139Q, P189S, G402L, A512T 305D10F, N135S, E139Q, P189S, E289Q, D411R, D422S, A512T 306 Q114L, N135S,E139Q, P189S, K225R, A367V, L503Q, Q508R, A512T 307 N135S, E139Q, P189S,G402V, A488Y, L503S, Q508A, A512T 308 N135S, E139Q, P189S, S245P, E289Q,G402S, A512T 309 N135S, E139Q, P189S, G402L, D411R, A488Y, L503S, A512T310 D10F, E88V, N135S, E139Q, P189S, K225R, E289Q, G402V, A488Y, L503R,A512T 311 N135S, E139Q, P189S, D422N, Q508A, A512T 312 D10F, N135S,E139Q, P189S, K225R, E289Q, G402A, L503S, Q508P, A512T 313 Q114L, N135S,E139Q, P189S, A488Y, L503S, Q508A, A512T 314 D10F, Q114L, N135S, E139Q,P189S, E289Q, G402L, A512T 315 Q114L, N135S, E139Q, P189S, L503Q, Q508R,A512T 316 N135S, E139Q, P189S, L405I, D411R, R509H, A512T 317 D10F,Q114L, N135S, E139Q, P189S, A488Y, L503A, A512T 318 N135S, E139Q, P189S,G402S, D411R, A512T 319 N135S, E139Q, P189S, G402V, A512T 320 D10F,Q114L, N135S, E139Q, P189S, G402A, D411R, A512T 321 D10F, Q114L, N135S,E139Q, P189S, G402V, Q508A, A512T 322 Q114L, N135S, E139Q, P189S, K225R,A488Y, L503R, A512T 323 Q114L, N135S, E139Q, P189S, D422S, A488Y, L503A,A512T 324 N135S, E139Q, P189S, T410V, A512T 325 S77K, N135S, E139Q,P189S, Y381R, L417L, Y501N, S502R, R509G, A512T 326 S77N, N135S, E139Q,P189S, Y381R, Y501N, S502R, A512T 327 N135S, E139Q, P189S, Y501Q, S502R,A512T 328 N135S, E139Q, P189S, L210N, Y381R, T410V, L417L, T431I, Y501N,S502R, A512T 329 S77R, N135S, E139Q, P189S, E304G, T431I, Y501Q, S502G,R509G, A512T 330 N135S, E139Q, P189S, L210N, E304G, R509G, A512T 331S77K, N135S, E139Q, P189S, E304G, Y381R, T431I, Y501N, A512T 332 S77N,N135S, E139Q, P189S, Y381R, L417L, Y501Q, R509G, A512T 333 N135S, E139Q,P189S, L417L, T431I, Y501G, S502G, A512T 334 N135S, E139Q, P189S, R509G,A512T 335 N135S, E139Q, P189S, E304G, A512T 336 S77R, N135S, E139Q,P189S, L417L, T431I, Y501N, S502R, R509G, A512T 337 N135S, E139Q, P189S,Y381R, L417L, T431I, Y501Q, S502G, R509G, A512T 338 N135S, E139Q, V186I,P189S, L417L, Y501Q, R509G, A512T 339 N135S, E139Q, L149E, P189S, E304G,Y381R, T431I, Y501Q, A512T 340 N135S, E139Q, L149E, P189S, Y501Q, S502R,A512T 341 N135S, E139Q, P189S, L417L, A512T 342 N135S, E139Q, P189S,L417L, R509G, A512T 343 N135S, E139Q, P189S, Y381R, A512T 344 S77K,N135S, E139Q, P189S, E304G, L417L, T431I, Y501Q, A512T 345 N135S, E139Q,P189S, Y381R, Y501Q, R509G, A512T 346 N135S, E139Q, L149E, P189S, E304G,Y381R, Y501N, A512T 347 S77K, N135S, E139Q, P189S, L417L, A512T 348N135S, E139Q, P189S, Y381R, T431I, Y501Q, A512T 349 N135S, E139Q, P189S,Y381R, L417V, R509G, A512T 350 N135S, E139Q, P189S, Y501Q, S502G, R509G,A512T 351 N135S, E139Q, P189S, L333S, P406F, A512T 352 N135S, E139Q,P189S, N419R, S434N, K511D, A512T 353 N135S, E139Q, P189S, P406L, A512T354 N135S, E139Q, P189S, P406A, N419R, K511Y, A512T 355 A2V, A108C,N135S, E139Q, P189S, T247A, L333S, P406G, N419V, K511D, A512T 356 N135S,E139Q, P189S, N419I, S434Y, K511Y, A512T 357 N135S, E139Q, P189S, P406A,N419V, K511S, A512T 358 N135S, E139Q, P189S, T247A, P406L, N419V, S434K,A512T 359 N135S, E139Q, P189S, N419I, K511S, A512T 360 A108C, N135S,E139Q, P189S, P406C, A506K, K511Y, A512T 361 N135S, E139Q, P189S, P406C,N419I, A506K, K511D, A512T 362 N135S, E139Q, P189S, L333S, P406F, N419I,K511D, A512T 363 N135S, E139Q, P189S, P406A, N419V, A512T 364 N135S,E139Q, P189S, P406L, N419R, S434H, K511D, A512T 365 N135S, E139Q, P189S,P406L, K511S, A512T 366 N135S, E139Q, P189S, S434H, K511D, A512T 367N135S, E139Q, P189S, P406G, N419I, S434N, K511Y, A512T 368 A2V, A108C,N135S, E139Q, P189S, L333S, P406V, N419R, A512T 369 N135S, E139Q, P189S,P406V, N419I, S434N, K511S, A512T 370 N135S, E139Q, P189S, P406V, N419V,A512T 371 A108L, N135S, E139Q, P189S, T247A, P406L, N419V, A506K, K511Y,A512T 372 A2V, A108W, N135S, E139Q, P189S, P406G, N419R, A512T 373N135S, E139Q, P189S, T247A, S434K, K511D, A512T 374 N135S, E139Q, P189S,P406A, A512T 375 A2V, G111D, N135S, E139Q, P189S, P406V, N419V, A512T376 E88G, N135S, E139Q, P189S, P406V, V413V, N419I, R509G, K510D, A512T377 N135S, E139Q, P189S, A296V, P406V, N419V, A512T 378 N135S, E139Q,P189S, L210N, P406V, N419V, L503S, R509H, K510H, A512T 379 N135S, E139Q,P189S, A371A, P406V, N419V, L503S, R509D, K510H, A512T 380 N135S, E139Q,P189S, P406V, N419V, R509H, K510D, A512T 381 N135S, E139Q, P189S, P406V,N419V, R509H, K510H, A512T 382 N135S, E139Q, P189S, P406V, N419V, R509G,K510N, A512T 383 N135S, E139Q, P189S, P406V, N419V, A512T 384 N135S,E139Q, P189S, P406W, N419V, R509H, K510D, A512T 385 H62R, N135S, E139Q,P189S, P406V, L417L, N419V, S434N, L503S, K510D, A512T 386 N135S, E139Q,P189S, P406V, N419V, K510D, A512T 387 N135S, E139Q, P189S, A296T, P406V,N419V, N459Q, R509H, K510D, A512T 388 N135S, E139Q, P189S, P406V, N419V,R509D, K510D, A512T 389 N135S, E139Q, P189S, L210N, P406V, N419V, A512T390 N135S, E139Q, P189S, P406V, N419V, L503S, R509D, A512T 391 N135S,E139Q, P189S, L210K, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 392N135S, E139Q, P189S, E304G, G402I, P406A, N419I, R509G, K510D, A512T 393N135S, E139Q, P189S, E304G, G402I, P406A, N419V, L503S, R509G, K510D,A512T 394 E115G, N135S, E139Q, P189S, E304G, P406V, N419V, L503S, R509G,K510D, A512T 395 N135S, E139Q, P189S, E304G, G402S, P406A, L417L, N419V,R509G, K510D, A512T 396 N135S, E139Q, P189S, G402S, P406A, N419V, L503S,K510H, A512T 397 N135S, E139Q, P189S, P406V, L417L, N419V, A512T 398N135S, E139Q, P189S, P406A, N419V, N459Q, R509G, K510H, A512T 399 N135S,E139Q, K163T, P189S, E304G, G402S, P406A, L417L, N419I, L503S, R509G,K510Y, A512T 400 N135S, E139Q, P189S, P406V, N419V, N459Q, R509G, A512T401 N135S, E139Q, P189S, G402V, P406A, V413V, N419I, L503S, R509G,K510H, A512T 402 N135S, E139Q, P189S, E304G, G402I, P406A, N419V, L503S,K510H, A512T 403 N135S, E139Q, P189S, E304G, P406V, V413V, N419I, K510D,A512T 404 N135S, E139Q, P189S, P406V, L417L, N419V, L503S, R509G, K510H,A512T 405 N135S, E139Q, P189S, E304G, P406V, N419V, R509G, K510D, A512T406 N135S, E139Q, P189S, P406V, N419V, L503S, R509G, K510D, A512T 407N135S, E139Q, P189S, G402S, P406L, V413V, L417L, N419V, L503S, K510H,A512T 408 N135S, E139Q, P189S, P406A, V413V, L417L, N419V, R509G, K510D,A512T 409 N135S, E139Q, P189S, P406V, N419V, L503S, R509G, K510H, A512T410 N135S, E139Q, P189S, P406V, N419I, R509G, K510D, A512T 411 N135S,E139Q, P189S, G402V, P406V, N419I, A506K, A512T 412 N135S, E139Q, P189S,P406V, N419V, A506K, A512T 413 N135S, E139Q, P189S, E304G, G402L, P406V,N419V, A506K, A512T 414 N135S, E139Q, P189S, E304G, G402V, P406L, N419I,A506K, A512T 415 K23R, N135S, E139Q, P189S, E304G, G402V, P406A, N419V,A512T 416 N135S, E139Q, P189S, G265S, P406V, N419V, Y501D, A506K, A512T417 N135S, E139Q, P189S, E304G, P406V, N419I, A512T 418 N135S, E139Q,P189S, E304G, G402L, P406C, N419V, A512T 419 N135S, E139Q, P189S, E304G,G402I, P406W, N419V, A512T 420 N135S, E139Q, P189S, E304G, G402L, P406V,N419I, A506K, A512T 421 N135S, E139Q, P189S, G402V, P406C, N419V, A512T422 N135S, E139Q, P189S, E304G, P406A, N419V, A506K, A512T 423 N135S,E139Q, P189S, P406V, N419I, A512T 424 N135S, E139Q, P189S, P406V, N419V,D430E, N459Q, L503S, R509D, K510D, A512T 425 N135S, E139Q, P189S, D213R,P406V, N419V, N459Q, A488S, L503S, R509D, K510D, A512T 426 N135S, E139Q,P189S, P340G, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 427 N135S,E139Q, P189S, P406V, N419V, N428K, N459Q, L503S, R509D, K510D, A512T 428N135S, E139Q, P189S, P406V, N419V, N459Q, G467Q, L503S, R509D, K510D,A512T 429 N135R, E139Q, P189S, P406V, N419V, N459Q, L503S, R509D, K510D,A512T 430 A92R, N135S, E139Q, P189S, P406V, N419V, N459Q, L503S, R509D,K510D, A512T 431 K15V, N135S, E139Q, P189S, P406V, N419V, N459Q, L503S,R509D, K510D, A512T 432 L70E, N135S, E139Q, P189S, P406V, N419V, N459Q,L503S, R509D, K510D, A512T 433 N135S, E139Q, P189S, P406V, T410W, N419V,N459Q, L503S, R509D, K510D, A512T 434 N135S, E139Q, P189S, S245H, P406V,N419V, N459Q, L503S, R509D, K510D, A512T 435 H99P, N135S, E139Q, P189S,P406V, N419V, N459Q, L503S, R509D, K510D, A512T 436 N135S, E139Q, P189S,H378K, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 437 N135S, E139Q,P189S, A390V, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 438 N135S,E139Q, P189S, P406V, N419V, S434K, N459Q, L503S, R509D, K510D, A512T 439N135S, E139Q, P189S, P406V, N419V, Q433Q, N459Q, L503S, R509D, K510D,A512T 440 N135S, E139Q, P189S, Y381K, P406V, N419V, N459Q, L503S, R509D,K510D, A512T 441 L70Q, N135S, E139Q, P189S, P406V, N419V, N459Q, L503S,R509D, K510D, A512T 442 N135S, E139Q, P189S, P406V, L407Y, N419V, N459Q,L503S, R509D, K510D, A512T 443 N135S, E139Q, P189S, A284F, P406V, N419V,N459Q, L503S, R509D, K510D, A512T 444 N135S, E139Q, P189S, P406V, D411N,N419V, N459Q, L503S, R509D, K510D, A512T 445 N135S, E139Q, I154I, P189S,P406V, N419V, N459Q, L503S, R509D, K510D, A512T 446 N135S, E139Q, P189S,P198P, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 447 N135S, E139Q,P189S, S245P, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 448 N135S,E139Q, N178R, P189S, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 449N135S, E139Q, P189S, S245G, P406V, N419V, N459Q, L503S, R509D, K510D,A512T 450 N135S, E139Q, P189S, V306I, P406V, N419V, Q433C, N459Q, L503S,R509D, K510D, A512T 451 R66Y, N135S, E139Q, P189S, P406V, N419V, N459Q,L503S, R509D, K510D, A512T 452 R66G, N135S, E139Q, P189S, P406V, N419V,N459Q, L503S, R509D, K510D, A512T 453 N135S, E139Q, P189S, P406V, V413M,N419V, N459Q, L503S, R509D, K510D, A512T 454 N135S, E139Q, I187A, P189S,P406V, N419V, N459Q, L503S, R509D, K510D, A512T 455 N135S, E139Q, P189S,P406V, N419I, N459Q, L503S, R509D, K510D, A512T 456 N135S, E139Q, P189S,R343L, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 457 N135S, E139Q,P189S, G341P, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 458 N135S,E139Q, P189S, P406V, N419V, T437D, N459Q, L503S, R509D, K510D, A512T 459N135S, E139Q, Q181H, P189S, P406V, N419V, E453N, N459Q, L503S, R509D,K510D, A512T 460 N135S, E139Q, P189S, A284E, P406V, N419V, N459Q, L503S,R509D, K510D, A512T 461 N135S, E139Q, P189S, T307W, A371I, P406V, N419V,N459Q, L503S, R509D, K510D, A512T 462 N135S, E139Q, P189S, P406V, D411A,N419V, N459Q, L503S, R509D, K510D, A512T 463 N135S, E139Q, P189S, P406V,D411R, N419V, N459Q, L503S, R509D, K510D, A512T 464 N135S, E139Q, P189S,P406V, T410Y, N419V, N459Q, L503S, R509D, K510D, A512T 465 N135S, E139Q,P189S, P406V, N419V, N459Q, Y501R, L503S, R509D, K510D, A512T 466 K15R,N135S, E139Q, P189S, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 467N135S, E139Q, P189S, L405A, P406V, N419V, N459Q, L503S, R509D, K510D,A512T 468 N135S, E139Q, P189S, P406V, N419V, D422P, N459Q, L503S, R509D,K510D, A512T 469 N135S, E139Q, P189S, A390M, P406V, N419V, N459Q, L503S,R509D, K510D, A512T 470 N135S, E139Q, P189S, E228R, P406V, N419V, N459Q,L503S, R509D, K510D, A512T 471 N135S, E139Q, N175C, P189S, P406V, N419V,N459Q, L503S, R509D, K510D, A512T 472 V105M, N135S, E139Q, P189S, P406V,N419V, N459Q, L503S, R509D, K510D, A512T 473 N135S, E139Q, P189S, G352C,P406V, N419V, N459Q, L503S, R509D, K510D, A512T 474 N135S, E139Q, P189S,Y381R, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 475 Q19I, N135S,E139Q, P189S, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 476 N135S,E139Q, P189S, P406V, N419V, N459Q, V500R, L503S, R509D, K510D, A512T 477N135S, E139Q, P189M, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 478N135S, E139Q, N178T, P189S, P406V, N419V, N459Q, L503S, R509D, K510D,A512T 479 N135S, E139Q, P189S, P406V, N419V, D430R, N459Q, L503S, R509D,K510D, A512T 480 N135S, E139Q, P189S, P406V, N419V, T437Q, N459Q, L503S,R509D, K510D, A512T 481 D64Y, N135S, E139Q, P189S, P406V, N419V, N459Q,L503S, R509D, K510D, A512T 482 N135S, E139Q, P189S, P406V, N419V, E453N,N459Q, L503S, R509D, K510D, A512T 483 N135S, E139Q, P189S, P406V, D411H,N419V, N459Q, L503S, R509D, K510D, A512T 484 N135S, E139Q, P189S, T267A,P406V, N419V, N459Q, L503S, R509D, K510D, A512T 485 V105I, N135S, E139Q,P189S, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 486 N135S, E139Q,P189S, A284M, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 487 N135S,E139Q, N178Q, P189S, P406V, D411C, N419V, N459Q, L503S, R509D, K510D,A512T 488 N135S, E139Q, P189S, A390I, P406V, N419V, N459Q, L503S, R509D,K510D, A512T 489 N135S, E139Q, P189S, P406V, N419V, N459Q, G467R, L503S,R509D, K510D, A512T 490 N135S, E139Q, P189S, P406V, N419V, E453A, N459Q,L503S, R509D, K510D, A512T 491 N135S, E139Q, P189S, S245F, P406V, N419V,N459Q, L503S, R509D, K510D, A512T 492 N135S, E139Q, P189S, P406V, T410V,N419V, N459Q, L480Q, L503S, R509D, K5100, A512T 493 N135S, E139Q, P189S,P406V, N419V, N459Q, G467E, L503S, R509D, K510D, A512T 494 N135S, E139Q,P189S, V319I, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 495 N135S,E139Q, P189S, A390L, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 496N135S, E139Q, P189A, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 497N135S, E139Q, P189S, E206G, P406V, N419V, N459Q, L503S, R509D, K510D,A512T 498 N135S, E139Q, P189S, P406V, N419V, T431H, N459Q, L503S, R509D,K510D, A512T 499 D64R, N135S, E139Q, P189S, P406V, N419V, N459Q, L503S,R509D, K510D, A512T 500 N135S, E139Q, P189S, P406V, N419V, N459Q, L503S,R509D, K510D, A512K 501 N135S, E139Q, P189S, P406V, N419V, N459Q, L475R,L503S, R509S, K510D, A512T 502 N135S, E139Q, P189S, T307H, P406V, N419V,N459Q, L503S, R509D, K510D, A512T 503 N135S, E139Q, P189S, D377P, P406V,N419V, N459Q, L503S, R509D, K510D, A512T 504 N135S, E139Q, P189S, Y381N,P406V, N419V, N459Q, L503S, R509D, K510D, A512T 505 N135S, E139Q, P189S,V399Y, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 506 D10D, N135S,E139Q, P189S, P406V, N419V, N459Q, A488T, Y501H, L503S, R509D, K510D,A512T 507 N135S, E139Q, I187G, P189S, P406V, N419V, N459Q, L503S, R509D,K510D, A512T 508 N129H, N135S, E139Q, P189S, P406V, N419V, N459Q, L503S,R509D, K510D, A512T 509 N135S, E139Q, P189S, E228T, P406V, N419V, N459Q,L503S, R509D, K510D, A512T 510 N135S, E139Q, P189S, V208L, P406V, N419V,N459Q, L503S, R509D, K510D, A512T 511 N135S, E139Q, P189S, L365I, P406V,N419V, N459Q, L503S, R509D, K510D, A512T 512 N135S, E139Q, P189S, A284T,P406V, N419V, N459Q, L503S, R509D, K510D, A512T 513 N135S, E139Q, P189S,S245A, P406V, L414R, N419V, N459Q, L503S, R509D, K510D, A512T 514 N135S,E139Q, P189S, P406V, N419V, N459Q, A488R, L503S, R509D, K510D, A512T 515N135S, E139Q, P189S, E228A, P406V, N419V, N459Q, L503S, R509D, K510D,A512T 516 N135S, E139Q, P189S, V218L, P406V, N419V, N459Q, L503S, R509D,K510D, A512T 517 N135S, E139Q, P189S, I400T, P406V, N419V, N459Q, L503S,R509D, K510D, A512T 518 H62R, N135S, E139Q, P189S, P406V, D411H, N419V,N459Q, L503S, R509D, K510D, A512T 519 N135S, E139Q, P189S, P406V, N419V,T431R, N459Q, L503S, R509D, K510D, A512T 520 N135S, E139Q, P189S, M366N,P406V, N419V, N459Q, L503S, R509D, K510D, A512T 521 N135S, E139Q, P189I,P406V, N419V, N459Q, L503S, R509D, K510D, A512T 522 N135S, E139Q, I187Y,P189S, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 523 N135S, E139Q,P189S, L227M, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 524 N135S,E139Q, P189S, K225R, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 525N135S, E139Q, P189S, E228H, P406V, N419V, N459Q, L503S, R509D, K510D,A512T 526 N135S, E139Q, P189S, G402C, P406V, N419V, N459Q, L503S, R509D,K510D, A512T 527 N135S, E139Q, P189S, P406V, N419V, N459Q, L475R, L503S,R509D, K510D, A512T 528 N135S, E139Q, P189S, R342K, P406V, N419V, S434K,N459Q, L503S, R509D, K510D, A512T 529 V105I, N135S, E139Q, P189S, L331V,P406V, N419V, S434K, N459Q, G467Q, L503S, R509D, K510D, A512T 530 N135S,E139Q, P189S, A284T, P406V, N419V, S434K, N459Q, L503S, R509D, K510D,A512T 531 N135S, E139Q, P189S, T307H, P406V, N419V, S434K, N459Q, L503S,R509D, K510D, A512T 532 R66G, N135S, E139Q, N175C, N178T, P189S, K225R,G352C, P406V, N419V, S434K, N459Q, L503S, R509D, K510D, A512T 533 R66G,N135S, E139Q, N175C, N178T, P189S, K225R, L405A, P406V, N419V, S434K,N459Q, L503S, R509D, K510D, A512T 534 D64R, R66G, N135S, E139Q, P189S,K225R, L227M, P406V, N419V, S434K, N459Q, L503S, R509D, K510D, A512T,A513S 535 Q19I, R66G, N135S, E139Q, P189S, K225R, P406V, N419V, S434K,N459Q, L503S, R509D, K510D, A512T 536 D64R, R66G, N135S, E139Q, N175C,P189S, L227M, P406V, N419V, S434K, N459Q, L503S, R509D, K510D, A512T 537R66G, N129H, N135S, E139Q, N175C, P189S, K225R, L227M, G352C, L405A,P406V, N419V, S434K, N459Q, L503S, R509D, K510D, A512T 538 D64R, R66G,N135S, E139Q, P189S, G352C, L405A, P406V, N419V, S434K, N459Q, A488R,L503S, R509D, K510D, A512T 539 N129H, N135S, E139Q, P189S, G352C, L405A,P406V, N419V, S434K, N459Q, A488R, L503S, R509D, K510D, A512T 540 Q19I,N135S, E139Q, N175C, P189S, K225R, L227M, G352C, P406V, N419V, S434K,N459Q, L503S, R509D, K510D, A512T 541 R66G, N129H, N135S, E139Q, N175C,N178T, P189S, P406V, L407Y, N419V, S434K, N459Q, L503S, R509D, K510D,A512T 542 N129H, N135S, E139Q, N178T, P189S, L227M, P406V, N419V, S434K,N459Q, L503S, R509D, K510D, A512T 543 Q19I, D64R, R66G, N135S, E139Q,N175C, P189S, K225R, L227M, P406V, L407Y, N419V, S434K, N459Q, L503S,R509D, K510D, A512T 544 D64R, R66G, N135S, E139Q, N175C, N178T, P189S,L405A, P406V, N419V, S434K, N459Q, L503S, R509D, K510D, A512T 545 N135S,E139Q, N175C, P189S, K225R, L227M, E228R, G352C, L405A, P406V, N419V,S434K, N459Q, L503S, R509D, K510D, A512T 546 Q19I, R66G, N135S, E139Q,N178T, P189S, K225R, L405A, P406V, L407Y, N419V, S434K, N459Q, L503S,R509D, K510D, A512T 547 N129H, N135S, E139Q, N175C, P189S, L227M, G352C,P406V, N419V, S434K, N459Q, L503S, R509D, K510D, A512T 548 Q19I, D64R,N135S, E139Q, N175C, P189S, G352C, P406V, N419V, S434K, N459Q, A488R,L503S, R509D, K510D, A512T 549 Q19I, R66G, N129H, N135S, E139Q, N175C,N178T, P189S, K225R, P406V, N419V, S434K, N459Q, L503S, R509D, K510D,A512T 550 Q19I, N129H, N135S, E139Q, N175C, N178T, P189S, P406V, N419V,S434K, N459Q, L503S, R509D, K510D, A512T 551 Q19I, D64R, R66G, N135S,E139Q, P189S, G352C, P406V, N419V, S434K, N459Q, L503S, R509D, K510D,A512T 552 N135S, E139Q, N175C, P189S, K225R, L227M, G352C, P406V, L407Y,N419V, S434K, N459Q, L503S, R509D, K510D, A512T 553 D64R, R66G, N135S,E139Q, N175C, P189S, K225R, L227M, P406V, N419V, S434K, N459Q, L503S,R509D, K510D, A512T 554 D64R, R66G, N135S, E139Q, N175C, N178T, P189S,L227M, P406V, L407Y, N419V, S434K, N459Q, A488R, L503S, R509D, K510D,A512T 555 Q19I, D64R, R66G, N129H, N135S, E139Q, P189S, P406V, N419V,S434K, N459Q, A488R, L503S, R509D, K510D, A512T 556 Q19I, N129H, N135S,E139Q, N175C, N178T, P189S, L227M, G352C, P406V, N419V, S434K, N459Q,L503S, R509D, K510D, A512T 557 R66G, N135S, E139Q, N175C, P189S, L227M,L405A, P406V, L407Y, N419V, S434K, N459Q, A488R, L503S, R509D, K510D,A512T 558 N135S, E139Q, P189S, V218L, P406V, N419V, S434K, N459Q, L503S,R509D, K510D, A512T 559 N135S, E139Q, P189S, A262D, P340G, Y381R, P406V,D411R, N419V, D430E, S434K, N459Q, L475R, L503S, R509D, K510D, A512T 560N135S, E139Q, P189S, Y381N, P406V, D411A, N419V, S434K, N459Q, L503S,R509D, K510D, A512T 561 N135S, E139Q, P189S, T267A, P340G, Y381N, P406V,N419V, S434K, N459Q, L503S, R509D, K510D, A512T 562 N135S, E139Q, P189S,V218L, P406V, T410Y, D411N, N419V, S434K, N459Q, L503S, R509D, K510D,A512T 563 N135S, E139Q, P189S, P406V, D411N, N419V, S434K, N459Q, L503S,R509D, K510D, A512T 564 N135S, E139Q, P189S, P406V, T410W, D411A, N419V,S434K, N459Q, L503S, R509D, K510D, A512K 565 N135S, E139Q, P189S, P340G,P406V, N419V, S434K, N459Q, L503S, R509D, K510D, A512T 566 N135S, E139Q,P189S, P406V, T410W, D411R, N419V, S434K, N459Q, L503S, R509D, K510D,A512T 567 L70E, N135S, E139Q, P189S, T267A, P406V, N419V, S434K, N459Q,L503S, R509D, K510D, A512T 568 N135S, E139Q, P189S, T267A, P406V, N419V,S434K, N459Q, L503S, R509D, K510D, A512T 569 L70E, N135S, E139Q, P189S,P406V, T410Y, N419V, D430E, S434K, N459Q, L503S, R509D, K510D, A512T 570L70E, N135S, E139Q, P189S, P406V, N419V, S434K, N459Q, L503S, R509D,K510D, A512T 571 N135S, E139Q, P189S, P340G, P406V, D411A, N419V, D430E,S434K, N459Q, L503S, R509D, K510D, A512T 572 N135S, E139Q, P189S, P340G,P406V, T410W, D411R, N419V, S434K, N459Q, L503S, R509D, K510D, A512T 573N135S, E139Q, P189S, P406V, D411A, N419V, S434K, N459Q, L503S, R509D,K510D, A512T 574 N135S, E139Q, P189S, V218L, T267A, P406V, N419V, S434K,N459Q, L503S, R509D, K510D, A512T 575 N135S, E139Q, P189S, I400T, G402C,P406V, N419V, Q433Q, S434K, N459Q, L503S, R509D, K510D, A512T 576 N135S,E139Q, N178R, P189S, I400T, P406V, N419V, Q433Q, S434K, N459Q, L503S,R509D, K510D, A512T 577 N135S, E139Q, N178R, P189S, I400T, G402C, P406V,N419V, Q433Q, S434K, N459Q, L503S, R509D, K510D, A512T 578 N135S, E139Q,P189S, I400T, P406V, N419V, S434K, N459Q, L503S, R509D, K510D, A512T 579N135S, E139Q, P189S, I400T, G402C, P406V, N419V, S434K, N459Q, L503S,R509D, K510D, A512T 580 N135S, E139Q, P189S, V399Y, G402C, P406V, N419V,S434K, N459Q, L503S, R509D, K510D, A512T 581 N135S, E139Q, P189S, V399Y,I400T, G402C, P406V, N419V, Q433Q, S434K, N459Q, L503S, R509D, K510D,A512T 582 N135S, E139Q, N178R, P189S, V399Y, I400T, P406V, N419V, Q433Q,S434K, N459Q, L503S, R509D, K510D, A512T 583 N135S, E139Q, P189S, V399Y,I400T, P406V, N419V, Q433Q, S434K, N459Q, L503S, R509D, K510D, A512T 584N135S, E139Q, N178R, P189S, V399Y, P406V, N419V, S434K, N459Q, L503S,R509D, K510D, A512T 585 N135S, E139Q, P189S, P406V, N419V, Q433Q, S434K,N459Q, L503S, R509D, K510D, A512T 586 N135S, E139Q, P189S, L365I, G402C,P406V, N419V, S434K, N459Q, L503S, R509D, K510D, A512T 587 N135S, E139Q,P189S, S245P, G402C, P406V, N419V, Q433Q, S434K, N459Q, L503S, R509D,K510D, A512T 588 N135S, E139Q, N178R, P189S, V399Y, P406V, N419V, Q433Q,S434K, N459Q, L503S, R509D, K510D, A512T 589 N135S, E139Q, P189S, V399Y,P406V, N419V, S434K, N459Q, L503S, R509D, K510D, A512T 590 N135S, E139Q,N178R, P189S, V399Y, G402C, P406V, N419V, Q433Q, S434K, N459Q, L503S,R509D, K510D, A512T 591 N135S, E139Q, N178R, P189S, P406V, N419V, S434K,N459Q, L503S, R509D, K510D, A512T 592 N135S, E139Q, P189S, A390M, P406V,N419V, N459Q, L503S, R509D, K510D, A512T 593 N135S, E139Q, P189S, D377P,P406V, N419V, N459Q, L503S, R509D, K510D, A512T 594 N135S, E139Q, P189S,A390I, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 595 N135S, E139Q,P189S, P406V, N419V, N459Q, L475R, L503S, R509D, K510D, A512T 596 N135R,E139Q, P189S, A284F, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 597N135S, E139Q, P189S, A284M, P406V, N419V, N459Q, L503S, R509D, K510D,A512T 598 N135S, E139Q, N178T, P189S, P406V, N419V, N459Q, L503S, R509D,K510D, A512T 599 N135S, E139Q, I187G, P189S, P406V, N419V, N459Q, L503S,R509D, K510D, A512T 600 N135S, E139Q, P189I, A390L, P406V, N419V, N459Q,L503S, R509D, K510D, A512T 601 N135S, E139Q, N1750, P189S, P406V, N419V,N459Q, Y501R, L503S, R509D, K510D, A512T 602 V105I, N135S, E139Q, P189S,P406V, N419V, N459Q, L503S, R509D, K510D, A512T 603 N135S, E139Q, P189I,P406V, N419V, N459Q, L503S, R509D, K510D, A512T 604 N135S, E139Q, P189S,S245H, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 605 N135S, E139Q,P189S, M366N, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 606 P63S,N135S, E139Q, P189S, S245A, P406V, L407Y, N419V, N459Q, L503S, R509D,K510D, A512T 607 N135S, E139Q, P189S, H378K, P406V, N419V, N459Q, L503S,R509D, K510D, A512T 608 N135S, E139Q, P189S, A284M, P406V, N419V, N459Q,Y501R, L503S, R509D, K510D, A512T 609 N135R, E139Q, P189S, I400T, P406V,N419V, N459Q, L503S, R509D, K510D, A512T 610 N135S, E139Q, P189S, A390V,P406V, N419V, N459Q, L503S, R509D, K510D, A512T 611 N135S, E139Q, P189S,D377P, P406V, N419V, E453G, N459Q, L503S, R509D, K510D, A512T 612 N135S,E139Q, N178Q, P189S, P406V, N419V, N459Q, Y501R, L503S, R509D, K510D,A512T 613 N135R, E139Q, P189S, P406V, N419V, N459Q, L503S, R509D, K510D,A512T 614 N135S, E139Q, N178Q, P189S, H378K, P406V, N419V, N459Q, L503S,R509D, K510D, A512T 615 N135S, E139Q, P189S, P406V, N419V, E453N, N459Q,L503S, R509D, K510D, A512T 616 N135S, E139Q, P189S, S245F, P406V, N419V,N459Q, Y501R, L503S, R509D, K510D, A512T 617 V6P, V105I, N135S, E139Q,P189S, P406V, N419V, N459Q, L503S, R509D, K510D, A512T 618 N135S, E139Q,P189S, R404S, P406V, N419V, N459Q, Y501R, L503S, R509D, K510D, A512T 619N135S, E139Q, P189S, A284T, P406V, N419V, N459Q, L503S, R509D, K510D,A512T 620 N135S, E139Q, P189S, L365I, P406V, N419V, N459Q, L503S, R509D,K510D, A512T 621 N135S, E139Q, P189S, A284F, P406V, N419V, N459Q, L503S,R509D, K510D, A512T 622 S13T, N135R, E139Q, P189S, P406V, N419V, N459Q,L503S, R509D, K510D, A512T 623 N135S, E139Q, P189S, P406V, N419V, N459Q,Y501R, L503S, R509D, K510D, A512T 624 N135S, E139Q, P189S, P406V, D411N,N419V, N459Q, L503S, R509D, K510D, A512T 625 N135S, E139Q, P189S, E228R,P406V, N419V, N459Q, L503S, R509D, K510D, A512T 626 N135S, E139Q, P189S,P406V, N419V, N459Q, G467Q, L503S, R509D, K510D, A512T 627 N135S, E139Q,P189S, V319I, P406V, N419V, N459Q, G467E, L503S, R509D, K510D, A512T 628N135S, E139Q, P189S, S245P, P406V, N419V, N459Q, L503S, R509D, K510D,A512T 629 N135S, E139Q, P189S, Y381K, P406V, N419V, N459Q, L503S, R509D,K510D, A512T

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

The entire contents of U.S. provisional application No. 61/221,934,filed Jun. 30, 2009, U.S. provisional application No. 61/315,380, filedMar. 18, 2010, U.S. patent application Ser. No. 12/825,939, filed onJun. 29, 2010 and published as US 2011/0000125, are hereby incorporatedby reference in their entireties for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

SEQUENCE LISTING SEQ ID NO: 1M. algicola DG893 FAR DNA (codon optimized)ATGGCTACTCAACAACAACAGAACGGTGCATCTGCATCCGGCGTCTTGGAACAACTTCGTGGAAAGCACGTTCTTATCACAGGTACTACCGGATTTTTGGGCAAAGTGGTTCTGGAAAAGTTGATTCGTACTGTTCCGGATATTGGAGGTATTCATCTGCTGATTCGTGGCAATAAACGTCATCCAGCCGCTCGTGAACGTTTCCTGAACGAAATTGCGTCCTCCTCCGTCTTCGAACGTTTGCGTCACGATGATAATGAAGCCTTCGAGACCTTCTTGGAAGAACGTGTTCACTGTATTACCGGTGAGGTTACTGAATCCCGTTTTGGTTTGACACCTGAACGTTTTCGTGCTTTGGCCGGTCAGGTTGACGCTTTTATTAACAGCGCTGCAAGCGTGAACTTTCGTGAGGAATTGGATAAAGCCCTGAAAATCAACACCTTGTGTCTTGAAAATGTTGCTGCTCTTGCAGAATTGAACTCCGCTATGGCGGTCATTCAGGTTTCCACTTGTTACGTTAACGGTAAAAACTCCGGTCAAATTACCGAATCCGTCATTAAACCTGCTGGCGAATCCATTCCCCGTTCCACTGACGGTTACTACGAGATCGAAGAATTGGTCCATCTGTTGCAAGACAAGATTTCCGATGTTAAAGCTCGTTACTCCGGCAAAGTTCTGGAGAAAAAATTGGTTGATTTGGGTATTCGTGAGGCCAATAATTACGGATGGTCCGACACCTACACATTCACCAAATGGTTGGGTGAACAACTGCTGATGAAGGCCTTGTCTGGTCGTTCTTTGACTATTGTGCGTCCCTCTATTATTGAGTCCGCTTTGGAAGAACCTTCCCCTGGTTGGATCGAAGGCGTTAAAGTTGCCGATGCCATTATCTTGGCTTATGCCCGTGAAAAAGTTAGCCTGTTCCCTGGAAAACGTTCCGGCATTATTGATGTTATTCCTGTCGATTTGGTTGCGAACTCCATCATCTTGTCTCTGGCTGAGGCGTTGTCTGGTTCTGGTCAACGTCGTATTTATCAATGTTGCAGCGGTGGTTCTAATCCAATCTCCCTGGGTAAGTTCATTGATTATTTGATGGCCGAGGCTAAGACCAACTATGCTGCCTACGATCAACTGTTTTATCGTCGTCCTACTAAACCTTTCGTCGCCGTGAACCGTAAATTGTTTGACGTTGTTGTTGGTGGTATGCGTGTTCCTCTTTCTATTGCCGGTAAAGCTATGCGTTTGGCTGGTCAAAATCGTGAGTTGAAAGTGCTTAAGAACCTTGATACGACCCGTTCCCTTGCAACCATTTTTGGCTTCTATACTGCTCCCGACTATATCTTCCGTAACGATAGCTTGATGGCCCTGGCTTCTCGTATGGGTGAATTGGATCGTGTTCTTTTCCCAGTTGATGCTCGTCAAATTGATTGGCAGTTGTACTTGTGTAAAATTCATTTGGGTGGTCTGAACCGTTACGCTTTGAAGGAACGTAAACTGTATTCTTTGCGTGCTGCTGATACTCGTAAAAAAGCTGCC TAASEQ ID NO: 2 M. algicola DG893 FAR polypeptideMATQQQQNGASASGVLEQLRGKHVLITGTTGFLGKVVLEKLIRTVPDIGGIHLLIRGNKRHPAARERFLNEIASSSVFERLRHDDNEAFETFLEERVHCITGEVTESRFGLTPERFRALAGQVDAFINSAASVNFREELDKALKINTLCLENVAALAELNSAMAVIQVSTCYVNGKNSGQITESVIKPAGESIPRSTDGYYEIEELVHLLQDKISDVKARYSGKVLEKKLVDLGIREANNYGWSDTYTFTKWLGEQLLMKALSGRSLTIVRPSIIESALEEPSPGWIEGVKVADAIILAYAREKVSLFPGKRSGIIDVIPVDLVANSIILSLAEALSGSGQRRIYQCCSGGSNPISLGKFIDYLMAEAKTNYAAYDQLFYRRPTKPFVAVNRKLFDVVVGGMRVPLSIAGKAMRLAGQNRELKVLKNLDTTRSLATIFGFYTAPDYIFRNDSLMALASRMGELDRVLFPVDARQIDWQLYLCKIHLGGLNRYALKERKLYSLRAADTRKKAASEQ ID NO: 3 M. algicola DG893 FAR DNA (codon optimized)ATGGCCACCCAGCAGCAGCAGAACGGTGCATCCGCTTCGGGCGTTCTGGAGCAGCTTAGAGGCAAGCATGTCTTGATTACCGGTACTACAGGATTTCTGGGAAAGGTGGTTCTGGAGAAGCTGATCCGAACCGTGCCTGACATCGGTGGTATTCATCTGCTGATTAGAGGCAACAAGAGACATCCTGCTGCCAGAGAAAGATTCTTGAACGAAATCGCCTCTTCCTCTGTGTTCGAGCGGCTTAGACATGACGACAACGAAGCCTTTGAGACTTTCCTGGAGGAGCGTGTGCACTGCATCACCGGAGAAGTGACCGAGTCGAGATTTGGCCTTACTCCTGAGCGGTTCCGAGCCCTTGCTGGCCAAGTGGATGCCTTCATCAATTCCGCCGCCTCTGTTAACTTCAGAGAGGAGCTGGACAAGGCACTCAAGATCAACACCCTGTGTCTGGAGAACGTGGCTGCTCTGGCCGAACTTAACTCCGCTATGGCAGTGATCCAAGTTTCCACCTGTTACGTGAACGGCAAGAACTCTGGACAGATCACCGAGTCCGTTATCAAGCCCGCTGGCGAATCCATCCCCAGATCCACAGATGGCTACTACGAGATCGAGGAGCTGGTCCACCTTCTGCAAGACAAGATCTCCGACGTGAAGGCTCGATACTCTGGCAAGGTGTTGGAGAAGAAGCTGGTGGACCTGGGCATCCGAGAGGCGAACAACTACGGCTGGTCTGACACCTACACCTTCACCAAATGGCTCGGAGAGCAGCTTCTGATGAAAGCTCTGTCCGGAAGATCCCTGACTATCGTGCGGCCTTCCATCATCGAGTCGGCTCTTGAAGAGCCTTCTCCAGGTTGGATCGAGGGCGTGAAGGTTGCTGACGCCATCATCCTTGCGTACGCCAGAGAGAAGGTTTCGTTGTTCCCCGGCAAGCGATCTGGCATCATCGACGTTATCCCCGTGGATCTGGTGGCCAACTCTATCATTCTCTCTCTTGCTGAAGCCCTTTCTGGATCTGGCCAGCGTAGAATCTACCAATGTTGTTCTGGCGGTTCTAACCCGATTTCTCTGGGCAAGTTCATCGACTACCTTATGGCCGAAGCCAAGACCAACTATGCTGCCTACGACCAGCTCTTCTACCGACGACCCACCAAGCCCTTCGTCGCTGTGAACCGAAAGCTGTTCGATGTTGTCGTGGGAGGAATGCGAGTGCCTCTTTCCATTGCTGGCAAGGCCATGAGATTGGCGGGTCAGAATCGAGAATTGAAGGTTCTCAAGAACCTTGACACTACTCGATCGCTCGCTACTATCTTTGGATTCTACACTGCTCCTGACTACATCTTCCGGAATGACTCTCTGATGGCTCTTGCTTCCCGAATGGGAGAACTCGATCGTGTGCTGTTCCCTGTTGACGCTCGACAGATCGACTGGCAGCTCTACTTGTGTAAGATCCACCTGGGCGGCCTGAACCGATATGCTCTGAAAGAACGAAAGCTGTACAGCCTTAGAGCCGCTGATACCCGAAAGAAGGCTGCT TAASEQ ID NO: 4 M. aquaeolei FAR DNA (codon optimized)ATGGCTATCCAGCAGGTTCATCACGCCGACACATCCTCCTCTAAAGTCCTGGGTCAACTTCGTGGTAAACGTGTCTTGATTACCGGCACTACTGGATTCTTGGGTAAAGTCGTCTTGGAACGTTTGATTCGTGCCGTTCCTGACATCGGTGCTATCTACCTGCTGATTCGTGGTAACAAGCGTCACCCGGATGCTCGTTCTCGTTTCTTGGAGGAGATTGCTACCTCCTCTGTCTTTGATCGTTTGCGTGAAGCTGATTCCGAAGGTTTCGATGCTTTCCTGGAAGAACGTATTCACTGTGTTACTGGTGAAGTTACTGAAGCTGGTTTCGGTATTGGTCAAGAGGACTATCGTAAGTTGGCCACCGAATTGGACGCAGTCATCAATTCTGCTGCCTCCGTCAACTTCCGTGAGGAGTTGGATAAGGCTCTGGCCATCAACACTCTGTGTTTGCGTAACATCGCTGGTATGGTGGATCTTAACCCTAAGCTGGCCGTTCTTCAAGTCTCTACGTGTTACGTCAACGGTATGAACTCTGGTCAAGTTACTGAATCCGTCATCAAACCAGCTGGTGAAGCTGTTCCTCGTTCTCCTGATGGATTCTACGAGATCGAGGAATTGGTTCGTCTGCTGCAAGACAAGATTGAAGACGTTCAAGCACGTTACTCTGGTAAGGTGTTGGAGCGTAAGTTGGTTGATTTGGGTATTCGTGAGGCTAATCGTTACGGTTGGTCTGATACATACACCTTCACGAAATGGTTGGGTGAACAACTTCTGATGAAAGCCTTGAATGGTCGTACCTTGACTATTCTGCGTCCTAGCATCATTGAATCTGCTTTGGAAGAACCAGCACCTGGTTGGATTGAAGGCGTGAAAGTTGCAGATGCGATCATCTTGGCTTATGCTCGTGAGAAGGTTACTTTGTTTCCGGGTAAACGTTCTGGTATCATTGATGTGATTCCTGTTGACTTGGTTGCCAATTCCATCATCTTGTCTTTGGCTGAGGCTCTGGGCGAACCTGGTCGTCGTCGTATCTACCAATGTTGTTCTGGTGGTGGTAATCCTATCTCCCTGGGCGAGTTCATTGATCACCTGATGGCTGAATCCAAAGCCAACTATGCCGCATACGATCATCTGTTCTACCGTCAACCCTCCAAGCCTTTCCTTGCTGTCAACCGTGCTTTGTTCGACTTGGTTATCTCTGGTGTCCGTCTGCCTTTGTCTTTGACCGACCGTGTCTTGAAGCTGCTGGGCAACTCCCGTGACCTGAAGATGCTGCGTAACCTGGATACTACGCAATCCCTGGCTACTATCTTTGGCTTCTACACAGCCCCCGACTACATCTTCCGTAATGACGAGTTGATGGCCCTGGCTAACCGTATGGGCGAGGTTGATAAGGGTTTGTTCCCCGTTGATGCTCGTCTGATTGATTGGGAATTGTACCTGCGTAAGATTCACCTGGCTGGTTTGAACCGTTACGCCTTGAAGGAGCGTAAGGTTTACTCTTTGAAGACAGCCCGTCAGCGTAAGAAGGCA GCTTAASEQ ID NO: 5 M. aquaeolei FAR polypeptideMAIQQVHHADTSSSKVLGQLRGKRVLITGTTGFLGKVVLERLIRAVPDIGAIYLLIRGNKRHPDARSRFLEEIATSSVFDRLREADSEGFDAFLEERIHCVTGEVTEAGFGIGQEDYRKLATELDAVINSAASVNFREELDKALAINTLCLRNIAGMVDLNPKLAVLQVSTCYVNGMNSGQVTESVIKPAGEAVPRSPDGFYEIEELVRLLQDKIEDVQARYSGKVLERKLVDLGIREANRYGWSDTYTFTKWLGEQLLMKALNGRTLTILRPSIIESALEEPAPGWIEGVKVADAIILAYAREKVTLFPGKRSGIIDVIPVDLVANSIILSLAEALGEPGRRRIYQCCSGGGNPISLGEFIDHLMAESKANYAAYDHLFYRQPSKPFLAVNRALFDLVISGVRLPLSLTDRVLKLLGNSRDLKMLRNLDTTQSLATIFGFYTAPDYIFRNDELMALANRMGEVDKGLFPVDARLIDWELYLRKIHLAGLNRYALKERKVYSLKTARQRKKA ASEQ ID NO: 6 FAR Maa variant 370 polypeptideMATQQQQNGASASGVLEQLRGKHVLITGTTGFLGKVVLEKLIRTVPDIGGIHLLIRGNKRHPAARERFLNEIASSSVFERLRHDDNEAFETFLEERVHCITGEVTESRFGLTPERFRALAGQVDAFINSAASVSFREQLDKALKINTLCLENVAALAELNSAMAVIQVSTCYVNGKNSGQITESVIKSAGESIPRSTDGYYEIEELVHLLQDKISDVKARYSGKVLEKKLVDLGIREANNYGWSDTYTFTKWLGEQLLMKALSGRSLTIVRPSIIESALEEPSPGWIEGVKVADAIILAYAREKVSLFPGKRSGIIDVIPVDLVANSIILSLAEALSGSGQRRIYQCCSGGSNPISLGKFIDYLMAEAKTNYAAYDQLFYRRPTKPFVAVNRKLFDVVVGGMRVVLSIAGKAMRLAGVNRELKVLKNLDTTRSLATIFGFYTAPDYIFRNDSLMALASRMGELDRVLFPVDARQIDWQLYLCKIHLGGLNRYALKERKLYSLRAADTRKKTASEQ ID NO: 7 FAR Maa variant 391 polypeptideMATQQQQNGASASGVLEQLRGKHVLITGTTGFLGKVVLEKLIRTVPDIGGIHLLIRGNKRHPAARERFLNEIASSSVFERLRHDDNEAFETFLEERVHCITGEVTESRFGLTPERFRALAGQVDAFINSAASVSFREQLDKALKINTLCLENVAALAELNSAMAVIQVSTCYVNGKNSGQITESVIKSAGESIPRSTDGYYEIEELVHLLQDKISDVKARYSGKVLEKKLVDLGIREANNYGWSDTYTFTKWLGEQLLMKALSGRSLTIVRPSIIESALEEPSPGWIEGVKVADAIILAYAREKVSLFPGKRSGIIDVIPVDLVANSIILSLAEALSGSGQRRIYQCCSGGSNPISLGKFIDYLMAEAKTNYAAYDQLFYRRPTKPFVAVNRKLFDVVVGGMRVVLSIAGKAMRLAGVNRELKVLKNLDTTRSLATIFGFYTAPDYIFRNDSLMALAQRMGELDRVLFPVDARQIDWQLYLCKIHLGGLNRYALKERKLYSSRAADTDDKTASEQ ID NO: 8 FAR Maa variant 436 polypeptideMATQQQQNGASASGVLEQLRGKHVLITGTTGFLGKVVLEKLIRTVPDIGGIHLLIRGNKRHPAARERFLNEIASSSVFERLRHDDNEAFETFLEERVHCITGEVTESRFGLTPERFRALAGQVDAFINSAASVSFREQLDKALKINTLCLENVAALAELNSAMAVIQVSTCYVNGKNSGQITESVIKSAGESIPRSTDGYYEIEELVHLLQDKISDVKARYSGKVLEKKLVDLGIREANNYGWSDTYTFTKWLGEQLLMKALSGRSLTIVRPSIIESALEEPSPGWIEGVKVADAIILAYAREKVSLFPGKRSGIIDVIPVDLVANSIILSLAEALSGSGQRRIYQCCSGGSNPISLGKFIDYLMAEAKTNYAAYDKLFYRRPTKPFVAVNRKLFDVVVGGMRVVLSIAGKAMRLAGVNRELKVLKNLDTTRSLATIFGFYTAPDYIFRNDSLMALAQRMGELDRVLFPVDARQIDWQLYLCKIHLGGLNRYALKERKLYSSRAADTDDKTASEQ ID NO: 9 FAR Maa variant 438 polypeptideMATQQQQNGASASGVLEQLRGKHVLITGTTGFLGKVVLEKLIRTVPDIGGIHLLIRGNKRHPAARERFLNEIASSSVFERLRHDDNEAFETFLEERVHCITGEVTESRFGLTPERFRALAGQVDAFINSAASVSFREQLDKALKINTLCLENVAALAELNSAMAVIQVSTCYVNGKNSGQITESVIKSAGESIPRSTDGYYEIEELVHLLQDKISDVKARYSGKVLEKKLVDLGIREANNYGWSDTYTFTKWLGEQLLMKALSGRSLTIVRPSIIESALEEPSPGWIEGVKVADAIILAYAREKVSLFPGKRSGIIDVIPVDLVANSIILSLAEALSGSGQRRIYQCCSGGSNPISLGKFIDYLMAEAKTNYAAYDQLFYRRPTKPFVAVNRKLFDVVVGGMRVVLSIAGKAMRLAGVNRELKVLKNLDTTRKLATIFGFYTAPDYIFRNDSLMALAQRMGELDRVLFPVDARQIDWQLYLCKIHLGGLNRYALKERKLYSSRAADTDDKTASEQ ID NO: 10 FAR Maa variant 547 polypeptideMATQQQQNGASASGVLEQLRGKHVLITGTTGFLGKVVLEKLIRTVPDIGGIHLLIRGNKRHPAARERFLNEIASSSVFERLRHDDNEAFETFLEERVHCITGEVTESRFGLTPERFRALAGQVDAFIHSAASVSFREQLDKALKINTLCLENVAALAELNSAMAVIQVSTCYVCGKNSGQITESVIKSAGESIPRSTDGYYEIEELVHLLQDKISDVKARYSGKVMEKKLVDLGIREANNYGWSDTYTFTKWLGEQLLMKALSGRSLTIVRPSIIESALEEPSPGWIEGVKVADAIILAYAREKVSLFPGKRSGIIDVIPVDLVANSIILSLAEALSGSGQRRIYQCCSGCSNPISLGKFIDYLMAEAKTNYAAYDQLFYRRPTKPFVAVNRKLFDVVVGGMRVVLSIAGKAMRLAGVNRELKVLKNLDTTRKLATIFGFYTAPDYIFRNDSLMALAQRMGELDRVLFPVDARQIDWQLYLCKIHLGGLNRYALKERKLYSSRAADTDDKTASEQ ID NO: 11 FAR Maa variant 555 polypeptideMATQQQQNGASASGVLEILRGKHVLITGTTGFLGKVVLEKLIRTVPDIGGIHLLIRGNKRHPRAGERFLNEIASSSVFERLRHDDNEAFETFLEERVHCITGEVTESRFGLTPERFRALAGQVDAFIHSAASVSFREQLDKALKINTLCLENVAALAELNSAMAVIQVSTCYVNGKNSGQITESVIKSAGESIPRSTDGYYEIEELVHLLQDKISDVKARYSGKVLEKKLVDLGIREANNYGWSDTYTFTKWLGEQLLMKALSGRSLTIVRPSIIESALEEPSPGWIEGVKVADAIILAYAREKVSLFPGKRSGIIDVIPVDLVANSIILSLAEALSGSGQRRIYQCCSGGSNPISLGKFIDYLMAEAKTNYAAYDQLFYRRPTKPFVAVNRKLFDVVVGGMRVVLSIAGKAMRLAGVNRELKVLKNLDTTRKLATIFGFYTAPDYIFRNDSLMALAQRMGELDRVLFPVDARQIDWQLYLCKIHLRGLNRYALKERKLYSSRAADTDDKTSEQ ID NO: 12 FAR Maa variant 556 polypeptideMATQQQQNGASASGVLEILRGKHVLITGTTGFLGKVVLEKLIRTVPDIGGIHLLIRGNKRHPAARERFLNEIASSSVFERLRHDDNEAFETFLEERVHCITGEVTESRFGLTPERFRALAGQVDAFIHSAASVSFREQLDKALKINTLCLENVAALAELNSAMAVIQVSTCYVCGKTSGQITESVIKSAGESIPRSTDGYYEIEELVHLLQDKISDVKARYSGKVMEKKLVDLGIREANNYGWSDTYTFTKWLGEQLLMKALSGRSLTIVRPSIIESALEEPSPGWIEGVKVADAIILAYAREKVSLFPGKRSGIIDVIPVDLVANSIILSLAEALSGSGQRRIYQCCSGCSNPISLGKFIDYLMAEAKTNYAAYDQLFYRRPTKPFVAVNRKLFDVVVGGMRVVLSIAGKAMRLAGVNRELKVLKNLDTTRKLATIFGFYTAPDYIFRNDSLMALAQRMGELDRVLFPVDARQIDWQLYLCKIHLGGLNRYALKERKLYSSRAADTDDKTASEQ ID NO: 13Marinobacter algicola DG893 (FAR Maa) wild-type cDNA sequenceATGGCAACACAGCAGCAACAAAACGGAGCGTCAGCGTCCGGTGTTCTTGAGCAACTACGTGGTAAACACGTGCTGATCACCGGCACCACCGGGTTTCTTGGTAAGGTGGTACTGGAAAAATTGATTCGCACGGTGCCGGATATTGGCGGGATCCATCTTCTTATCCGTGGTAACAAAAGGCATCCTGCAGCACGGGAACGATTCCTCAACGAGATCGCCAGTTCTTCCGTGTTCGAACGCCTTCGGCACGATGACAACGAGGCGTTTGAAACCTTTCTTGAGGAACGCGTTCACTGCATCACCGGCGAAGTGACAGAGTCGCGTTTCGGGCTCACGCCGGAGCGGTTCCGTGCACTTGCCGGGCAGGTCGATGCGTTTATAAATTCCGCAGCCAGTGTGAACTTCCGGGAGGAACTCGACAAGGCGCTGAAGATTAACACCCTGTGCCTGGAGAACGTTGCCGCTCTGGCGGAGCTCAATAGCGCCATGGCGGTTATCCAGGTGTCCACCTGCTACGTCAATGGCAAGAATTCCGGCCAGATCACGGAGTCCGTCATCAAGCCGGCGGGCGAGTCTATTCCCCGCAGCACCGACGGCTACTATGAAATCGAAGAGCTTGTGCATTTGCTGCAGGACAAAATTTCCGACGTGAAAGCCCGATACTCCGGCAAAGTACTTGAAAAAAAGCTGGTGGACCTGGGGATTCGAGAGGCCAACAACTACGGCTGGAGTGACACCTACACGTTTACCAAATGGCTGGGTGAGCAACTCCTGATGAAAGCCCTTTCCGGGCGTTCACTTACGATTGTTCGCCCTTCCATCATTGAAAGTGCACTGGAAGAGCCTTCGCCAGGATGGATTGAAGGTGTGAAGGTGGCAGACGCCATTATCCTTGCCTATGCCCGTGAGAAGGTCTCCCTGTTCCCAGGCAAGCGTAGCGGCATTATCGATGTGATCCCGGTGGACCTGGTGGCCAACAGTATCATCTTGTCCCTGGCAGAAGCCCTTTCCGGGTCAGGGCAGCGCCGCATCTATCAATGCTGCAGTGGCGGTTCTAATCCGATTTCGCTGGGCAAGTTCATTGACTACCTGATGGCCGAAGCCAAGACCAACTATGCAGCGTATGACCAGTTGTTCTACCGACGGCCCACGAAACCGTTTGTGGCGGTCAATCGCAAGCTGTTTGATGTTGTGGTTGGCGGCATGCGCGTGCCGTTGTCGATTGCTGGCAAGGCAATGAGGCTGGCTGGCCAGAACCGTGAGCTCAAGGTTCTCAAAAACCTCGATACCACGCGTTCACTGGCCACCATCTTTGGTTTCTACACGGCACCGGATTACATCTTCCGTAACGATTCGCTGATGGCCCTGGCTTCGCGCATGGGTGAACTGGACCGTGTCCTGTTCCCGGTGGATGCGCGTCAGATTGACTGGCAGCTGTACTTGTGCAAGATCCACCTGGGAGGTCTCAACCGCTACGCTCTGAAGGAGCGAAAACTGTACAGCCTGCGGGCCGCCGACACCCGCAAAAAAGCCGCCSEQ ID NO: 14Marinobacter aquaeolei VT8 (FAR Maq) wild-type cDNA sequenceATGGCAATACAGCAGGTACATCACGCTGACACTTCATCATCAAAGGTGCTCGGACAGCTCCGTGGCAAGCGGGTTCTGATCACCGGTACCACTGGCTTTCTGGGCAAGGTGGTCCTCGAAAGGCTGATTCGGGCGGTGCCTGATATCGGCGCAATTTACCTGCTGATCCGGGGCAATAAACGGCATCCGGATGCTCGTTCCCGTTTCCTGGAAGAAATTGCCACCTCCTCGGTGTTTGACCGTCTTCGCGAGGCCGATTCAGAGGGATTTGACGCCTTTCTGGAAGAGCGCATTCACTGCGTGACCGGTGAGGTGACCGAAGCGGGTTTCGGGATAGGGCAGGAAGACTATCGCAAACTCGCCACCGAACTGGATGCGGTGATCAACTCCGCTGCAAGCGTGAATTTCCGTGAAGAGCTCGACAAGGCGCTGGCCATCAACACCCTGTGCCTTCGGAATATTGCCGGCATGGTGGATTTGAATCCGAAGCTTGCGGTCCTGCAGGTCTCCACCTGCTATGTCAATGGCATGAACTCGGGGCAGGTAACCGAATCGGTGATCAAGCCGGCAGGCGAGGCCGTGCCGCGTTCCCCGGACGGCTTCTATGAGATAGAAGAGCTTGTTCGCCTGCTTCAGGATAAAATTGAAGACGTTCAGGCCCGTTATTCCGGCAAAGTGCTGGAGAGGAAGCTGGTGGACCTGGGGATTCGGGAAGCCAACCGCTATGGCTGGAGCGATACCTACACCTTTACCAAGTGGCTGGGCGAACAGTTGCTGATGAAGGCGTTAAACGGGCGCACGCTGACCATTCTGCGTCCTTCGATTATCGAAAGTGCCCTGGAGGAACCAGCGCCCGGCTGGATTGAGGGGGTGAAGGTGGCAGATGCCATCATCCTGGCTTACGCACGGGAAAAAGTCACCCTCTTCCCGGGCAAACGCTCCGGTATCATCGATGTGATTCCAGTGGACCTGGTGGCCAACTCCATCATCCTTTCCCTGGCGGAAGCTCTTGGAGAACCCGGTCGACGTCGCATCTATCAATGTTGCAGCGGGGGCGGCAATCCAATCTCCCTGGGTGAGTTCATCGATCATCTCATGGCGGAATCAAAAGCCAATTACGCTGCCTACGATCACCTGTTCTACCGGCAGCCCAGCAAGCCGTTTCTGGCGGTTAACCGGGCGCTGTTTGATTTGGTGATCAGTGGTGTTCGCTTACCGCTCTCCCTGACGGACCGTGTGCTCAAATTACTGGGAAATTCCCGGGACCTGAAAATGCTCAGGAATCTGGATACCACCCAGTCGCTGGCAACCATTTTTGGTTTCTACACCGCGCCGGATTATATCTTCCGGAACGATGAGCTGATGGCGCTGGCGAACCGGATGGGTGAGGTCGATAAAGGGCTGTTCCCGGTGGATGCCCGCCTGATTGACTGGGAGCTCTACCTGCGCAAGATTCACCTGGCCGGGCTCAATCGCTATGCCCTGAAAGAACGAAAGGTGTACAGTCTGAAAACCGCGCGCCAGCGCAAAAAAGCT GCC

What is claimed is:
 1. A fatty alcohol forming acyl-CoA reductase (FAR)variant that has at least 90% sequence identity to SEQ ID NO:5, whereinthe variant comprises a substitution at one or more positions selectedfrom position 512, position 2, position 508, position 511, position 503,position 434, position 501, position 459, position 135, and position139, wherein the position is numbered with reference to SEQ ID NO:5, andwherein a cell in which the FAR variant is expressed produces more fattyalcohol than a corresponding cell of the same type in which thewild-type FAR from which the FAR variant is derived is expressed,wherein the cell is a yeast cell or a bacterial cell.
 2. The FAR variantof claim 1, wherein the cell is a bacterial cell.
 3. The FAR variant ofclaim 1, wherein the wild-type FAR is Marinobacter aquaeolei FAR havingthe amino acid sequence of SEQ ID NO:5.
 4. A recombinant polynucleotideencoding the FAR variant of claim
 1. 5. The polynucleotide of claim 4operably linked to a promoter.
 6. An expression vector comprising therecombinant polynucleotide of claim
 4. 7. A host cell comprising therecombinant polynucleotide of claim 4, wherein the host cell is a yeastcell or a bacterial cell.
 8. A host cell comprising a recombinantpolynucleotide sequence encoding a fatty alcohol forming acyl-CoAreductase (FAR) variant that has at least 90% sequence identity to SEQID NO:5, wherein the host cell is a yeast cell or a bacterial cell andwherein the variant comprises a substitution at one or more positionsselected from position 512, position 2, position 508, position 511,position 503, position 434, position 501, position 459, position 135,and position 139, wherein the position is numbered with reference to SEQID NO:5.
 9. The host cell of claim 8 that is E. coli.
 10. The host cellof claim 8, wherein the FAR variant comprises at least 95% sequenceidentity to SEQ ID NO:5.
 11. The host cell of claim 10, wherein the FARvariant protein has 100% identity to SEQ ID NO:5 except for thesubstitutions present in any individual FAR variant selected fromvariant numbers 1-629 in Table
 5. 12. The host cell of claim 8, whereinthe variant comprises a substitution at one or both of positions 512 and2, and wherein: (a) the amino acid at position 512 is glycine, lysine,proline, glutamine, serine, or threonine; or (b) the amino acid atposition 2 is glycine, proline, threonine, phenylalanine, or glutamine.13. The host cell of claim 12, wherein the FAR variant further comprisesa substitution at one or both of positions 508 and 511, wherein: (a) theamino acid at position 508 is glycine or serine; or (b) the amino acidat position 511 is aspartic acid, glycine, or proline.
 14. The host cellof claim 12, wherein the FAR variant further comprises a substitution atone or more of positions 503, 434, and 501, and wherein: (a) the aminoacid at position 503 is glutamine, arginine, or serine; (b) the aminoacid at position 434 is phenylalanine, lysine, or tryptophan; or (c) theamino acid at position 501 is glycine, asparagine, proline, glutamine,serine, or tryptophan.
 15. The host cell of claim 12, wherein the FARvariant further comprises a substitution at one or more of positions459, 135, and 139, wherein: (a) the amino acid at position 459 isglycine; (b) the amino acid at position 135 is lysine; or (c) the aminoacid at position 139 is glutamine.
 16. The host cell of claim 8 thatproduces at least 1.5 times more fatty alcohol than a corresponding cellof the same type expressing a wild-type FAR from which the FAR variantis derived.
 17. The host cell of claim 8, wherein: (a) at least 30% ofthe fatty alcohol produced is C12-C14 fatty alcohols; or (b) at least55% of the fatty alcohol produced is C16-C18 fatty alcohols; or (c) atleast 90% of the fatty alcohol produced is C14-C18 fatty alcohols. 18.The host cell of claim 8 that produces a fatty alcohol profilecomprising an increased amount of C14:0 (1-tetradecanol) fatty alcoholand a decreased amount of C18:1 (cis Δ¹¹-1-octadecenol) fatty alcoholrelative to a corresponding cell of the same type expressing a wild-typeFAR from which the FAR variant is derived.
 19. The host cell of claim 8,wherein at least 5 g/L of recoverable fatty alcohols are produced.
 20. Amethod of producing fatty alcohols, the method comprising culturing thehost cell of claim 8 in a culture medium under conditions in which thefatty alcohols are produced.
 21. A method of producing a detergentcomposition, the method comprising: combining the fatty alcoholsproduced by the method of claim 20, or a fraction thereof, with adetergent component selected from sodium carbonate, a complexationagent, zeolites, a protease, a lipase, amylase, carboxymethyl cellulose,optical brighteners, colorants and perfumes, thereby producing thedetergent composition.
 22. A method of producing a compositioncomprising alkanes or alkenes, the method comprising reducing fattyalcohols produced by the method of claim 20, or a fraction thereof, toproduce alkanes or alkenes.
 23. A method of producing a compositioncomprising fatty esters, the method comprising modifying fatty alcoholsproduced by the method of claim 20, or a fraction thereof, to producefatty esters.
 24. A method of producing a fuel composition, the methodcomprising reducing or esterifying the fatty alcohols produced by themethod of claim 20, or a fraction thereof, to yield the fuelcomposition.
 25. A method of producing fuel comprising (a) producingfatty alcohols according to the method of claim 20, and (b) subjectingthe fatty alcohols, or a fraction thereof, to one or more chemicalreactions to generate alkanes, whereby fuel is produced.
 26. The methodof claim 25 wherein the fatty alcohols comprise at least 90% C14-C18fatty alcohols and optionally contain less than 1% C18:0 fatty alcohols.27. An E. coli cell comprising a recombinant polynucleotide sequenceencoding a fatty alcohol forming acyl-CoA reductase (FAR) variant thathas at least 90% sequence identity to SEQ ID NO:5, wherein the FARvariant comprises a substitution at one or more positions selected fromposition 512, position 2, position 508, position 511, position 503,position 434, position 501, position 459, position 135, and position139, wherein the position is numbered with reference to SEQ ID NO:5. 28.The cell of claim 27 that produces more fatty alcohol than acorresponding cell of the same type in which a FAR polypeptide having100% identity to SEQ ID NO:5 is expressed.
 29. A composition comprising:(a) fatty alcohols produced by the method of claim 20; or (b) a fattyalcohol derivative prepared by subjecting the fatty alcohols in (a), ora fraction thereof, to one or more chemical reactions that produce afatty alcohol derivative.